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Information on EC 1.14.13.25 - methane monooxygenase (soluble) and Organism(s) Methylosinus trichosporium and UniProt Accession P27355
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1.14.13.25 methane monooxygenase (soluble)IUBMB Comments
The enzyme is soluble, in contrast to the particulate enzyme, EC 1.14.18.3. Broad specificity; many alkanes can be hydroxylated, and alkenes are converted into the corresponding epoxides; CO is oxidized to CO2, ammonia is oxidized to hydroxylamine, and some aromatic compounds and cyclic alkanes can also be hydroxylated, but more slowly.
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Word Map
- 1.14.13.25
- methanotrophs
- methanol
- methylosinus
- capsulatus
- methylococcus
- trichosporium
-
methane-oxidizing
- methylocystis
- ch4
- methylomonas
- dioxygen
-
dinuclear
- methylobacter
- trichloroethylene
- methylomicrobium
- alkane
-
diironii
-
ammonia-oxidizing
-
landfill
-
upland
-
antiferromagnetically
-
wetland
-
high-valent
-
non-motile
-
copper-containing
-
carboxylate-bridged
-
peat
-
copper-dependent
-
dicopper
- ch3oh
- nitrosomonas
-
cometabolic
-
diferrous
-
methanobactins
- energy production
-
mixed-valent
-
gammaproteobacterial
- sphagnum
-
t-rflp
-
seep
- propene
- synthesis
- biotechnology
- degradation
-
exafs
-
nitrify
-
peroxo
The taxonomic range for the selected organisms is: Methylosinus trichosporium
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
particulate methane monooxygenase, soluble methane monooxygenase, methane mono-oxygenase, methane monooxygenase hydroxylase, soluble methane monooxygenase hydroxylase, methane hydroxylase, cytoplasmic methane monooxygenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
methane hydroxylase
-
-
-
-
methane mono-oxygenase
-
-
-
-
MMOB
-
cofactor-free, monomeric component of sMMO, has regulatory role in catalysis
MMOH
-
hydroxylase component of sMMO, contains a binuclear nonheme iron active site that is essential for O2 activation and subsequent methane oxidation
MMOR
-
reductase component of sMMO, contains a FAD cofactor and a [2Fe-2S] cluster, is responsible for transferring the reducing equivalents from NAD(P)H to MMOH
oxygenase, methane mono-
-
-
-
-
pMMO
sMMO
soluble methane monooxygenase
sMMO
-
soluble methane monooxygenase
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
methane + NAD(P)H + H+ + O2 = methanol + NAD(P)+ + H2O
mechanism
-
methane + NAD(P)H + H+ + O2 = methanol + NAD(P)+ + H2O
kinetic model of protein component interaction
-
methane + NAD(P)H + H+ + O2 = methanol + NAD(P)+ + H2O
reaction cycle, via formation of the hydroperoxo intermediate compound P and the key reaction cycle intermediate compound Q, overview
-
methane + NAD(P)H + H+ + O2 = methanol + NAD(P)+ + H2O
reaction mechanism, substrate radical intermediates in the reaction of soluble methane monooxygenase, the reaction involves the conversion of the enzyme hydroxylase component, MMOH, diiron cluster to a strongly oxidizing bis-l-oxo-Fe(IV)2 species termed compound Q, that is capable of the fissure of stable CH bonds in methane and many other hydrocarbons
-
methane + NAD(P)H + H+ + O2 = methanol + NAD(P)+ + H2O
catalyzes the oxidation of methane through the activation of O2 at a nonheme biferrous center in the hydroxylase component MMOH
-
methane + NAD(P)H + H+ + O2 = methanol + NAD(P)+ + H2O
reaction mechanism with transition states, MMOHred to MMOHox transitions for open cores of MMOHQ, detailed overview. The reactivity of the Fe(IV)=O unit is modulated by the ligand to the second iron
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
-
-
-
-
oxidation
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -
SYSTEMATIC NAME
IUBMB Comments
methane,NAD(P)H:oxygen oxidoreductase (hydroxylating)
The enzyme is soluble, in contrast to the particulate enzyme, EC 1.14.18.3. Broad specificity; many alkanes can be hydroxylated, and alkenes are converted into the corresponding epoxides; CO is oxidized to CO2, ammonia is oxidized to hydroxylamine, and some aromatic compounds and cyclic alkanes can also be hydroxylated, but more slowly.
CAS REGISTRY NUMBER
COMMENTARY
51961-97-8
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
1-butene + NAD(P)H + O2
1,2-epoxybutane + NAD(P)+ + H2O
-
-
-
?
biphenyl + NAD(P)H + H+ + O2
2-hydroxybiphenyl + 4-hydroxybiphenyl + NAD(P)+ + H2O
-
-
-
-
?
butane + NAD(P)H + O2
1-butanol + 2-butanol + NAD(P)+ + H2O
-
-
-
-
?
cis-2-butene + NAD(P)H + O2
cis-2,3-epoxybutane + cis-2-buten-1-ol + 2-butanone + NAD(P)+ + H2O
-
-
-
?
cyclohexane + NAD(P)H + O2
cyclohexanol + NAD(P)+ + H2O
-
-
-
?
cyclohexene + NAD(P)H + O2
epoxycyclohexane + 2-cyclohexen-1-ol + NAD(P)+ + H2O
-
-
-
?
dimethyl ether + NAD(P)H + O2
methanol + formaldehyde + NAD(P)+ + H2O
-
no activity
-
-
?
ethylbenzene + NAD(P)H + H+ + O2
1-phenylethanol + 3-ethylphenol + 4-ethylphenol + NAD(P)+ + H2O
-
-
-
-
?
isobutane + NAD(P)H + O2
2-methyl-1-propanol + 2-methyl-2-propanol + NADP+ + H2O
-
-
-
?
isopentane + NAD(P)H + O2
2-methylbutan-1-ol + 3-methylbutan-1-ol + 2-methylbutan-2-ol + 3-methylbutan-2-ol + NADP+ + H2O
-
-
-
?
methane + trans-dichloroethylene + vinyl chloride + trichloroethylene + ?
formaldehyde + ?
-
each of these compounds is completely degraded by sMMO-expressing cells when initial concentrations are either 0.01 or 0.03 mM
-
-
?
naphthalene + NAD(P)H + H+ + O2
alpha-naphthol + beta-naphthol + NAD(P)+ + H2O
-
-
-
-
?
naphthalene + NAD(P)H + O2
alpha-naphthol + beta-naphthol + NAD(P)+ + H2O
-
oxidized by sMMO
-
-
?
naphthalene + NADH + H+
alpha-naphthol + beta-naphthol + NAD+ + H2O
-
-
-
-
?
propylene + duroquinol + O2
propylene oxide + reduced duroquinol + H2O
-
-
-
-
?
propylene + NADH + H+ + O2
propylene epoxide + NAD+ + H2O
-
-
-
-
?
toluene + NAD(P)H + H+ + O2
benzyl alcohol + cresol + NAD(P)+ + H2O
-
-
-
-
?
toluene + NAD(P)H + H+ + O2
benzyl alcohol + NAD(P)+ + H2O
-
-
-
?
trans-2-butene + NAD(P)H + O2
trans-2,3-epoxybutane + trans-2-buten-1-ol + NAD(P)+ + H2O
-
-
-
?
methane + NAD(P)H + H+ + O2
methanol + NAD(P)+ + H2O
-
-
-
-
?
methane + NAD(P)H + H+ + O2
methanol + NAD(P)+ + H2O
-
-
-
?
methane + NAD(P)H + H+ + O2
methanol + NAD(P)+ + H2O
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
-
-
-
?
methane + NAD(P)H + H+ + O2
methanol + NAD(P)+ + H2O
methane hydroxylation through methane monooxygenases is a key aspect due to their control of the carbon cycle in the ecology system
-
-
?
methane + NAD(P)H + H+ + O2
methanol + NAD(P)+ + H2O
presentation of experimental and computational data consistent with an open-core structure for the key intermediate in methane oxidation
-
-
?
methane + NAD(P)H + O2
methanol + NAD(P)+ + H2O
-
-
-
-
?
methane + NADH + H+ + O2
methanol + NAD+ + H2O
-
-
-
?
nitrobenzene + NADH + O2
nitrophenol + NAD+ + H2O
-
an electron is removed from nitrobenzene by Q in the first step of the reaction and then the bound hydroxyl radical formed in this process rebounds to form nitrophenol
-
-
?
propane + NAD(P)H + O2
1-propanol + 2-propanol + NAD(P)+ + H2O
-
-
-
-
?
propane + NAD(P)H + O2
1-propanol + 2-propanol + NAD(P)+ + H2O
-
-
-
?
propene + NAD(P)H + O2
1,2-epoxypropane + NAD(P)+ + H2O
-
-
-
?
propene + NAD(P)H + O2
1,2-epoxypropane + NAD(P)+ + H2O
-
-
-
?
additional information
?
-
-
oxidation of deuterated compounds
-
-
?
additional information
?
-
-
effects of spin-traps on MMO activity, overview
-
-
?
additional information
?
-
-
inactive toward anthracene and phenanthrene
-
-
?
additional information
?
-
-
pMMO has broader substrate specificity but lower activity with smaller hydrocarbons like methane, ethane, and propene compared to pMMO
-
-
?
additional information
?
-
-
naphthalene assay for sMMO activity
-
-
?
additional information
?
-
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
a colorimetric assay is adopted for the sMMO activity detection of biofilm
-
-
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
methane + NAD(P)H + O2
methanol + NAD(P)+ + H2O
-
-
-
-
?
methane + NAD(P)H + H+ + O2
methanol + NAD(P)+ + H2O
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
-
-
-
?
methane + NAD(P)H + H+ + O2
methanol + NAD(P)+ + H2O
methane hydroxylation through methane monooxygenases is a key aspect due to their control of the carbon cycle in the ecology system
-
-
?
methane + NADH + H+ + O2
methanol + NAD+ + H2O
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cytochrome c
-
one of three protein components is a soluble CO-binding cytochrome c
[2Fe-2S]-center
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
in the 2Fe-2S cluster-containing reductase (MMOR)
FAD
-
protein C, reductase component: contains 1 mol FAD per mol protein
FAD
soluble methane monooxygenase consists of three subunits: a hydroxylase bridged with binuclear iron cluster, an NADH-dependent reductase component containing both flavin adenine dinucleotide (FAD) and ferredoxin [Fe2S2] cofactors, and regulatory protein which controls the reaction between the previous two. Low-temperature activation of methane is primarily achieved via Fe/Fe complex in the hydroxylase subunit. The Fe2S2 complex in soluble methane monooxygenase reductase only acts as a wired mediator to assist electron transport from the NAD/FAD redox couple to the di-iron complex in the hydroxylase. NAD and FAD simultaneously bind to a canyon region located midway between the two lobes in the reductase, forming a continuous wire, assisting the electron transport. The regulatory protein plays a vital role in helping the hydroxylase and reductase subunits to interface and causing conformational changes that control methane oxidation
NADH
-
-
438921, 438922, 438924, 438932, 438933, 438934, 438935, 438941, 438946, 438948, 657998, 671721, 672102, 672130, 684566, 685262, 685272, 701759, 744100
NADH
soluble methane monooxygenase consists of three subunits: a hydroxylase bridged with binuclear iron cluster, an NADH-dependent reductase component containing both flavin adenine dinucleotide (FAD) and ferredoxin [Fe2S2] cofactors, and regulatory protein which controls the reaction between the previous two. Low-temperature activation of methane is primarily achieved via Fe/Fe complex in the hydroxylase subunit. The Fe2S2 complex in soluble methane monooxygenase reductase only acts as a wired mediator to assist electron transport from the NAD/FAD redox couple to the di-iron complex in the hydroxylase. NAD and FAD simultaneously bind to a canyon region located midway between the two lobes in the reductase, forming a continuous wire, assisting the electron transport. The regulatory protein plays a vital role in helping the hydroxylase and reductase subunits to interface and causing conformational changes that control methane oxidation
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Cu2+
Fe
Fe2+
Iron
Zn2+
-
the purified pMMO contains 1.3 Zn2+ ions per 100 kDa protomer, the membrane-bound pMMO contains 2.7 Zn2+ ions per 100 kDa protomer
additional information
Cu2+
-
copper-containing protein component contains one copper atom per molecule
Cu2+
-
cytochrome component contains 0.3-0.8 atoms copper per molecule
Cu2+
-
the membrane-bound pMMO contains 4.8 Cu2+ ions per 100 kDa protomer the purified pMMO contains 1.4 Cu2+ ions per 100 kDa protomer, the enzyme contains a dinuclear copper center
Cu2+
-
cells adapted to the respective medium, either lacking Cu (sMMO production) or containing 0.01 mM Cu (pMMO production)
Fe
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
the enzyme uses a nonheme, oxygen-bridged dinuclear iron cluster in the active site
Fe
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
the sMMOH active site contains a dinuclear iron cluster, which serves to activate molecular oxygen for insertion into the C-H bond of methane
Fe2+
-
the purified pMMO contains 7.6 Fe2+ ions per 100 kDa protomer, the membrane-bound pMMO contains 2.1 Fe2+ ions per 100 kDa protomer
Iron
-
non-heme iron, 3.02 mol iron per mol of enzyme, addition of exogenous FeCl2 or FeCl3 does not affect the enzyme activity
Iron
soluble methane monooxygenase consists of three subunits: a hydroxylase bridged with binuclear iron cluster, an NADH-dependent reductase component containing both flavin adenine dinucleotide (FAD) and ferredoxin [Fe2S2] cofactors, and regulatory protein which controls the reaction between the previous two. Low-temperature activation of methane is primarily achieved via Fe/Fe complex in the hydroxylase subunit. The Fe2S2 complex in soluble methane monooxygenase reductase only acts as a wired mediator to assist electron transport from the NAD/FAD redox couple to the di-iron complex in the hydroxylase. NAD and FAD simultaneously bind to a canyon region located midway between the two lobes in the reductase, forming a continuous wire, assisting the electron transport. The regulatory protein plays a vital role in helping the hydroxylase and reductase subunits to interface and causing conformational changes that control methane oxidation
additional information
-
the enzyme contains very low or no amounts of copper and zinc
additional information
-
sMMO activity and expression does not require Cu2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
poly-beta-hydroxybutyrate
-
MMO activity dramatically decreases when cellular poly-beta-hydroxybutyrate accumulates in the second stage. The more poly-beta-hydroxybutyrate are accumulated, the slower MMO activity decreases. Cellular poly-beta-hydroxybutyrate content has some influence on the maintenance of the MMO activity
2,4-Dichloro-(6-phenylphenoxy)ethylamine hydrochloride
-
no inhibition
additional information
-
different chlorinated hydrocarbons cause different inhibition patterns
-
additional information
-
when cultivated in nutrients deficiency culture, activity of sMMO in strain OB3b decreases, whereas activity of sMMO in strain IMV3011 does not decrease
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
steady-state kinetics
-
additional information
additional information
-
effects of the C-terminal region of the B component MMOB and the active site diiron cluster of sMMO on the steady-state turnover, stopped-flow transient kinetics of the reaction cycle of wild-type and mutant enzymes, overview, deuterium kinetic isotope effect for the reaction of Q with methane, overview
-
additional information
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
impact of the incorporation of 5-fluorotryptophan and BTFA on sMMO steady state and single-turnover kinetics
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
turnover numbers of component A
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.00011
-
purified enzyme, using propylene and duroquinol as substrates
0.0029
-
membrane-bound enzyme, using propylene and NADH as substrates
0.003
-
membrane-bound enzyme, using propylene and duroquinol as substrates
additional information
additional information
-
effects of spin-traps on MMO activity, spin trapping by 5,5-dimethyl-1-pyrroline N-oxide and alpha-4-pyridyl-1-oxide N-tert-butylnitrone, mechanism, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.4 - 7.4
-
pH 6.4: about 20% of activity maximum, pH 7.4: about 25% of activity maximum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
438921, 438922, 438924, 438932, 438933, 438934, 438935, 438941, 438946, 438948, 671477, 672102, 672130, 672690, 684566, 685262, 685272, 701759, 744100
-
-
P27353 (alpha/MmoX), P27355 (gamma/MmoZ), P27354 (beta/MmoY), P27356 (MmoB), Q53563 (MmoC), Q53562 (MmoD). The soluble methane monooxygenase (sMMO) consists of four components A/MMOH (composed of alpha/MmoX, beta/MmoY and gamma/MmoZ), B/MMOB (MmoB), C/MMOR (MmoC) and D/MMOD (MmoD)
SwissProt
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
-
enzyme from cells grown under conditions of low copper availability
-
membrane-bound particulate enzyme form termed pMMO
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
mutations in the core region of MMOB and in the N- and C-termini cause dramatic changes in the rate constants for steps in the sMMOH reaction cycle
metabolism
physiological function
additional information
metabolism
ammonia-supplied Methylosinus trichosporium OB3b containing soluble methane monooxygenase (sMMO) grow at the fastest rate, while the highest poly-beta-hydroxybutyrate content is obtained by transferring nitrate-supplied bacteria with the expression of particulate methane monooxygenase (pMMO) to nitrogen-free mineral salts (NFMS) + 0.005 mmol/l Cu medium
metabolism
methane hydroxylation through methane monooxygenases is a key aspect due to their control of the carbon cycle in the ecology system
physiological function
-
the enzyme is strongly regulated by the availability of copper. Methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain
physiological function
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
full activity of soluble methane monooxygenase (sMMO) depends upon the formation of a 1:1 complex of the regulatory protein MMOB with each alpha subunit of the (alphabetagamma)2 hydroxylase, sMMOH
physiological function
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the conversion of methane to methanol at ambient temperature and pressure. The enzyme consists of three protein components: a 245 kDa (alphabetagamma)2 hydroxylase (sMMOH), a 38 kDa flavin adenine dinucleotide (FAD) and 2Fe-2S cluster-containing reductase (MMOR), and a 15 kDa cofactorless regulatory component (MMOB). The sMMOH active site contains a dinuclear iron cluster, which serves to activate molecular oxygen for insertion into the C-H bond of methane. The resting state of sMMOH contains a diferric cluster (Fe3+Fe3+, sMMOHox) in which the irons are bridged by two solvent (OH- or H2O) molecules in addition to the carboxylate of Glu144. sMMOHox can form a complex with MMOR and receive two electrons to form the diferrous cluster (Fe2+Fe2+, sMMOHred) in which Glu243 shifts to bridge the irons via one carboxylate oxygen, one bridging solvent is lost, and the bond to the second solvent is weakened. In this new configuration, the diiron cluster can bind O2 between the irons upon dissociation of the weakly bound solvent. But O2 binding is observed to be very slow in the absence of the regulatory component MMOB. Binding of MMOB effects a 1000fold increase in the rate constant for the O2 binding to the diiron cluster to form the first spectroscopically distinct intermediate of the reaction cycle, termed P*. One cause of the decreased rate of O2 binding in the sMMOH active site in the absence of MMOB is the near closure of the molecular tunnel that mediates the transit of O2 from the solvent. This bottleneck is relieved by conformational changes in both MMOB and sMMOHred when the sMMOHred:MMOB complex forms. A second cause of the low reactivity of O2 with sMMOHred is the position of the Glu209 ligand to the diiron cluster, which blocks the approach to the open iron coordination site. An angle change of this residue in the sMMOHred:MMOB complex exposes the site for O2 binding. The formation of intermediate P* is followed by a spontaneous formation of a peroxo-intermediate P, and finally, O-O bond cleavage to yield the reactive dinuclear Fe4+ intermediate Q. Q can react directly with methane to form methanol with the incorporation of one atom of oxygen sourced from O2. Intermediate Q is generated and stabilized by precisely coordinated sMMO protein component interactions. Regulation of electron transfer in the sMMO system, mechanism, modeling, detailed overview. MMOR causes both the N-terminal tail and the core region of MMOB to dissociate from sMMOH
physiological function
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. The diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. Although chemically reduced sMMOH can carry out the oxygenation chemistry alone, the reaction only proceeds at a physiologically relevant rate when sMMOH is complexed with MMOB. Many regulatory functions of MMOB have been discovered but its most important effects are to decrease the redox potential of the diiron cluster by 132 mV, accelerate O2 binding by 1000fold, increase the turnover number 150fold, and tune sMMOH to selectively bind and oxygenate methane over other more easily oxidized hydrocarbons
physiological function
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
the metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O2, protons, and CH4 to an oxygen-bridged diferrous metal cluster located in the buried active site
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
enzyme sMMO structural reorganization through subunits MMOH and MMOB including the dinuclear iron cluster, detailed overview
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
exogenous ligands bound to the diiron cluster of the sMMOH:MMOB complex induce conformational changes, structural analysis, overview. Bottlenecks between cavities are regulated by flexible residues. Bottleneck regulated by residues V105, F109, V285, and L289 is located between cavities 3 and 2. Cavity 2 is separated from the active site cavity 1 by another bottleneck controlled by residues L110, F188, L216, F282 and F286. Cavities 3 and 2 are connected in all of the Mt sMMOH and sMMOH:MMOB crystal structures. The MMOB binding-induced reorganization of the bottleneck residues L216 and L110 in sMMOH serves to isolate cavity 1 from cavity 2 in the complex. The pore is located between helices E and F and has been proposed to be involved in regulating the access of substrates and release of products (CH3OH) to and from the active site, respectively. The strictly conserved amino acids T213, N214, and E240 are considered the pore gating residues that regulate these processes. The pore is a uniquely polar region on the sMMOH surface as it is flanked by hydrophobic amino acids A210, V218, L237, L244, and M247 on helices E and F. The side chain of T213 lines the active site cavity and the hydroxyl moiety points towards the diiron cluster. Chemical reduction of the diiron cluster causes the middle of helix E to twist, resulting in T213 and N214 to shift 2.2 A and 3.2 A, respectively. The rotameric conformations of the hydrophobic residues V218, L244, and M247 are altered as well, helping to create a chemical environment that does not favor stable binding of water molecules to the region around the pore. MMOB binding to sMMOH causes structural rearrangement of the pore residues as well. The side chain of E240 is no longer solvent exposed, and instead, traverses the width of the Pore. This new conformation blocks the access of substrates through the Pore into the active site cavity. The side chain of T213 is shifted 2.2 A compared to its position in Mt sMMOHox and rotated about 180° compared to its position in Mt sMMOHred. This new conformation positions the side chain hydroxyl moiety of T213 to face away from the diiron cluster and form a hydrogen bond with E240. MMOB covers the pore while in complex with sMMOH, further limiting access to the active site by this route
additional information
-
exogenous ligands bound to the diiron cluster of the sMMOH:MMOB complex induce conformational changes, structural analysis, overview. Bottlenecks between cavities are regulated by flexible residues. Bottleneck regulated by residues V105, F109, V285, and L289 is located between cavities 3 and 2. Cavity 2 is separated from the active site cavity 1 by another bottleneck controlled by residues L110, F188, L216, F282 and F286. Cavities 3 and 2 are connected in all of the Mt sMMOH and sMMOH:MMOB crystal structures. The MMOB binding-induced reorganization of the bottleneck residues L216 and L110 in sMMOH serves to isolate cavity 1 from cavity 2 in the complex. The pore is located between helices E and F and has been proposed to be involved in regulating the access of substrates and release of products (CH3OH) to and from the active site, respectively. The strictly conserved amino acids T213, N214, and E240 are considered the pore gating residues that regulate these processes. The pore is a uniquely polar region on the sMMOH surface as it is flanked by hydrophobic amino acids A210, V218, L237, L244, and M247 on helices E and F. The side chain of T213 lines the active site cavity and the hydroxyl moiety points towards the diiron cluster. Chemical reduction of the diiron cluster causes the middle of helix E to twist, resulting in T213 and N214 to shift 2.2 A and 3.2 A, respectively. The rotameric conformations of the hydrophobic residues V218, L244, and M247 are altered as well, helping to create a chemical environment that does not favor stable binding of water molecules to the region around the pore. MMOB binding to sMMOH causes structural rearrangement of the pore residues as well. The side chain of E240 is no longer solvent exposed, and instead, traverses the width of the Pore. This new conformation blocks the access of substrates through the Pore into the active site cavity. The side chain of T213 is shifted 2.2 A compared to its position in Mt sMMOHox and rotated about 180° compared to its position in Mt sMMOHred. This new conformation positions the side chain hydroxyl moiety of T213 to face away from the diiron cluster and form a hydrogen bond with E240. MMOB covers the pore while in complex with sMMOH, further limiting access to the active site by this route
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
significant conformational changes must be imparted within sMMOH by the binding of MMOB. Small-molecule tunnel analysis, overview
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
soluble methane monooxygenase component interactions monitored by 19F NMR spectroscopy. Modeling for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
structure-spectroscopy correlations for intermediate Q of soluble methane monooxygenase, QM/MM calculations. Modeling of the MMOH oxidative and reductive state, Moessbauer parameters and electronic structure of MMOHox. The selection of plausible models include the following: (1) bis-mu-oxo bridged cores, (2) mu-OepsilonGlu243 bridged diamond cores, inspired from the MMOHred structure, as suggested recently, (3) mu-oxo bridged open cores, and (4) mu-OepsilonGlu243 bridged open cores. Closed- and open-core conformations for the key intermediate in sMMO. Optimized cores of eight MOHQ models are analyzed for molecular structure and electronic structure
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
15800
-
component B containing FAD and [Fe2-S2]-cluster, gel filtration
22700
-
component A: 2 * 54400 alpha, 2 * 43000 beta + 2 * 22700 gamma, sedimentation velocity, SDS-PAGE, amino acid analysis
23000
-
2 * 58000, alpha-subunit, + 2 * 36000, beta-subunit, + 2 * 23000, gamma-subunit, (alphabetagamma)2, SDS-PAGE
245000
36000
-
2 * 58000, alpha-subunit, + 2 * 36000, beta-subunit, + 2 * 23000, gamma-subunit, (alphabetagamma)2, SDS-PAGE
43000
-
component A: 2 * 54400 alpha, 2 * 43000 beta + 2 * 22700 gamma, sedimentation velocity, SDS-PAGE, amino acid analysis
54400
-
component A: 2 * 54400 alpha, 2 * 43000 beta + 2 * 22700 gamma, sedimentation velocity, SDS-PAGE, amino acid analysis
58000
-
2 * 58000, alpha-subunit, + 2 * 36000, beta-subunit, + 2 * 23000, gamma-subunit, (alphabetagamma)2, SDS-PAGE
additional information
additional information
-
see under subunits: molecular weights of the subunits of components
additional information
-
see under subunits: molecular weights of the subunits of components
additional information
-
see under subunits: molecular weights of the subunits of components
additional information
-
3 components: 1 soluble CO-binding cytochrome c, 1 copper-containing protein, and 1 small protein, SDS-PAGE
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexamer
-
2 * 58000, alpha-subunit, + 2 * 36000, beta-subunit, + 2 * 23000, gamma-subunit, (alphabetagamma)2, SDS-PAGE
trimer
additional information
?
-
component A: 2 * 54400 alpha, 2 * 43000 beta + 2 * 22700 gamma, sedimentation velocity, SDS-PAGE, amino acid analysis
trimer
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
1 * 245000, alpha-subunitMMOH, + 1 * 37000, beta-subunit MMOR, + 1 * 15000, gamma-subunit MMOB
trimer
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme, all three sMMO protein components are: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB), (alphabetagamma)2, 1 * 245000, hydroxylase (sMMOH), + 1 * 38000, flavin adenine dinucleotide (FAD) and 2Fe-2S cluster-containing reductase (MMOR) + 1 * 1000 cofactorless regulatory component (MMOB)
additional information
-
see under molecular weight for the size of the protein components
additional information
-
see under molecular weight for the size of the protein components
additional information
-
see under molecular weight for the size of the protein components
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
enzyme system consists of 3 protein components A, B, C
additional information
-
3 components: 1 soluble CO-binding cytochrome c, 1 copper-containing protein, and 1 small protein, SDS-PAGE
additional information
-
interaction of the soluble methane monooxygenase regulatory component, MMOB, and the active site-bearing hydroxylase component, MMOH, spin labeling with 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy, high affinity of labeled MMOB for the oxidized MMOH decreases substantially with increasing pH and increasing ionic strength but is nearly unaffected by addition of nonionic detergents, the MMOB-MMOH complex is stabilized by electrostatic interactions, overview
additional information
-
the enzyme mainly exists of alpha-helical regions, circular dichroism measurement
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
the sMMO enzyme consists of three protein components: a 245 kDa (alphabetagamma)2 hydroxylase (MMOH), a 37 kDa FAD and Fe2S2 cluster-containing reductase (MMOR), and a 15 kDa regulatory protein (MMOB). The active site is buried deep within sMMOH and contains an oxygen-bridged dinuclear FeIII cluster in which the irons are bridged by two hydroxo moieties and a carboxylate from Glu144. After reduction, the diiron cluster functions to activate O2 and insert an oxygen atom into a highly stable (105 kcal/mol bond dissociation energy) C-H bond of methane. Although chemically reduced sMMOH can carry out the oxygenation chemistry alone, the reaction only proceeds at a physiologically relevant rate when sMMOH is complexed with MMOB. Reduction of the diiron cluster causes a shift in the position of Glu243, a monodentate ligand to Fe2 in the diferric cluster. In the shifted position, Glu243 bridges Fe1 and Fe2 via one of its carboxylate oxygens, thereby directly displacing one of the bridging solvents. The second bridging solvent bond is weakened, which presumably allows facile displacement by O2 to begin the oxygen activation process
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
analysis of the cyrstal structure of the sMMOH:5FWMMOB complex (PDB ID 7M8Q) showing the interface region containing 5FW76 and 5FW77, and of the structure of the sMMOH:BTFA-K15C-5FW-MMOB complex (PDB ID 7M8R) interface region showing the relative position of the BTFA and 5FW 19F-labels
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
crystallization of wild-type and mutant enzyme complexes by sitting drop vapour diffusion method, for the sMMOH:DBL2 and sMMOH:H5A proteins, 0.0015 ml of protein solution containing 0.06 mM sMMOH and 0.12 mM of either DBL2 or H5A in 100 mM MOPS buffer, pH 7.0, with 0.0015 ml cyrstallization solution containing 20% PEG 3350 and 0.2 M Na2HPO4, pH 8.8, equilibration against 0.5 ml of crystallization solution, 2-3 days at room temperature. For the sMMOH:DBL1 and sMMOH:H33A proteins, 0.0015 ml of protein solution containing 0.06 mM sMMOH and 0.12 mM of either DBL1 or H33A in 100 mM MOPS buffer, pH 7.0, with 0.0015 ml cyrstallization solution containing 21% PEG 3350 and 0.2 M Na2HPO4, pH 6.6, equilibration against 0.5 ml of crystallization solution, 2-3 days at room temperature, X-ray diffraction structure determination and analysis at 1.82-2.40 A resolution, structure modeling
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
diferric and diferrous states of both sMMOH and the sMMOH:MMOB complexes, X-ray diffraction structure determination and analysis at 1.52-2.35 A resolution, the structures are analyzed for O2 access routes enhanced when the complex forms. Evaluation of the structures of oxidized, diferric Mt sMMOH (sMMOHox, PDB:6VK6, 1.52 A), chemically reduced diferrous Mt sMMOH (sMMOHred, PDB:6VK7, 2.12 A), Mt sMMOHox:MMOB (form 1, PDB ID 6VK5, 1.86 A, and form 2, PDB ID 6VK8, 2.03 A), and Mt sMMOHred:MMOB (PDB ID 6VK4, 2.35 A). The two alphabetagamma protomers of sMMOH protein in the crystal are related by 2fold crystallographic symmetry
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
sitting drop vapour diffusion method with 100 mM cacodylate, pH 6.5, 20% (v/v) PEG 3000, 250 mM magnesium formate or MnCl2
-
sMMOH:MMOB complex in fully oxidized and fully reduced states, sitting drop vapor diffusion method, mixing of 0.0225 ml of protein solution containing 0.053 mM sMMOH and 0.106 mM MMOB in 25 mM MOPS, pH 7.0, with 0.0075 ml of reservoir solution containing 100 mM HEPES/MOPS, pH 7.5, 30 mM NaI, 30 mM NaBr, 30 mM NaF, 20% v/v glycerol, 10% w/v PEG 4000, and 10 mM FeCl3, equilibration against 0.5 ml reservoir solution, 3 days at room temperature, X-ray diffraction structure determination and analysis at 1.95-2.9 A resolution. Microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b are serially exposed to X-ray free electron laser (XFEL) pulses, where the 35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Structural reorganization analysis. The position of Glu243 relative to Fe2 is shifted to the bridging position. Detailed method overview
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A115C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
A62C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview, the mutant MMOH-MMOB complex is perturbed by salts but not nonionic detergents
D71C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
D87C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
G119C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
H33A
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis, an N-terminal region variant, structure analysis
H5A
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis, an N-terminal region variant, structure analysis
K15C
K44C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
L110C
-
mutant shows inverted or shifted regioselectivity with naphthalene, biphenyl, and ethylbenzene as a substrate compared to the wild type enzyme
L110G
-
mutant shows inverted or shifted regioselectivity with naphthalene, biphenyl, and ethylbenzene as a substrate compared to the wild type enzyme
L110R
-
mutant shows inverted or shifted regioselectivity with naphthalene, biphenyl, and ethylbenzene as a substrate compared to the wild type enzyme
L110Y
-
mutant shows inverted or shifted regioselectivity with naphthalene, biphenyl and ethylbenzene as a substrate compared to the wild type enzyme
N107G/S109A/S110A/T111A
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis, mutations in the core region, termed the Quad variant, structure analysis
N107G/S110A
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis, a binary derivative of the Quad variant, termed DBL1. The DBL1 mutation in MMOB leads to a loss of the S110 hydrogen bond with N214 of the sMMOH alpha-subunit, structure analysis
R133C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
S109A/T111A
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis, a binary derivative of the Quad variant, termed DBL2, structure analysis
S109C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
T111C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
T111Y
V39C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
V39F
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis in the MMOB component, the mutant component variant nearly halts the reaction of the reconstituted sMMO system
V39R
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis in the MMOB component, the mutant component variant nearly halts the reaction of the reconstituted sMMO system
V41E
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis in the MMOB component, the mutant component variant nearly halts the reaction of the reconstituted sMMO system
V41F
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis in the MMOB component, the mutant component variant nearly halts the reaction of the reconstituted sMMO system
V41R
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
site-directed mutagenesis in the MMOB component, the mutant component variant nearly halts the reaction of the reconstituted sMMO system
V68C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
Y102C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
additional information
K15C
-
site-directed mutagenesis of enzyme component MMOH, mobility and accessibility parameters for the spin-labeled MMOB mutants alone and in complex with MMOH in comparison to the wild-type enzyme, overview
T111Y
-
mutant enzyme with increased rate constant for the reaction of large substrates such as ethane, furan, and nitrobenzene with the reactive MMOH (regulatory componant of the enzyme) intermediate Q while decreasing the rate constant for the reaction with methane. The regiospecificity for nitrobenzene oxidation is altered and 10fold more T111Y than wild-type MMOB is required to maximize the rate of turnover
T111Y
-
the T111Y variant of MMOB causes only a small increase in reactivity
additional information
-
construction of deletion mutants of subunit MMOB missing 5, 8, and 13 C-terminal residues, the mutations cause progressive decreases in the maximum steady-state turnover number, as well as lower apparent rate constants for formation of the key reaction cycle intermediate compound Q, the deletions result in substantial uncoupling at or before the P intermediate due to competition between slow H2O2 release from one of the intermediates and the reaction that carries this intermediate on to the next step in the cycle, which is slowed by the mutation
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
construction of N- and C-terminal truncation variants. The dramatic effects of specific mutations on specific steps of the reaction cycle include the (i) retarding O2-binding (MMOB DELTA2-29 and V41R), (ii) uncoupling the O2-activation reaction from hydrocarbon oxidation (MMOB DELTA126-138), (iii) relaxing the size-selective entry of hydrocarbon substrates (Quad, DBL2), (iv) disrupting the quantum-tunneling nature of the HAT reaction of Q with methane (Quad), and (v) significantly increasing or decreasing the rate constants of reaction cycle steps (H33A, DBL1, DELTA126-138, H5A, V41R, and V39R). Effect of the MMOB variants on small-molecule access tunnels, overview
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
establishing of a methanotrophic co-metabolic system, built in the gcSBBR seeded by soil at a ventilation opening of coal mine in the presence of Cu2+, and seeded with sMMO in Methylosinus trichosporium OB3b and particulate methane monooxygenase (pMMO) in Methylocystis parvus in a biofilm, microbial community analyses and microbial 16S rRNA sequencing, and postulated pathway of methanotrophic co-metabolic system, overview. Six kinds of methanotrophs are detected from the seeded soil, namely, Methylocystis (0.29%), Methylocystaceae_unclassified (0.16%), Methylocystaceae_uncultured (0.04%) and Methylocaldum (0.14%), Methylococcaceae_unclassified (0.10%), Methylobacteriaceae_uncultured (0.04%), totally accounting for 0.77%
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
kinetic analysis of MMOB mutants, overview
additional information
-
kinetic analysis of MMOB mutants, overview
additional information
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFAK15C-5FW-MMOB are readily detected at 5 microM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
instability of the enzyme during purification is probably due to loss of iron
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
3 components: a soluble CO-binding cytochrome c, a copper-containing protein, another small protein
-
native enzyme 8.08fold by anion exchange chromatography, ultrafiltration, gel filtration, and again anion exchange chromatography, to 95% purity
-
recombinant enzyme component MMOB from Escherichia coli by solubilization from the 12000 x g pellet with 8 M urea, followed by dilution for enzyme refolding, and gel filtration
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene mmoX, quantitative RT-PCR enzyme expression analysis
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
mutants are expressed in sMMO-negative cells of Methylosinus trichosporium
-
plasmids encoding wild-type MMOB and W77F and K15C MMOB are transformed into Escherichia coli BL21(DE3) chemically competent cells
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
quantitative RT-PCR expression analysis of genes pmoA, mmoX, and mbnA
-
recombinant enzyme expression in Escherichia coli strain BL21(DE3)
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
substantial improvements to the system for mutagenesis of soluble methane monooxygenase and expression of recombinant enzymes in the methanotroph Methylosinus trichosporium OB3b expression system. This system can be utilised to make a number of new mutants and to engineer soluble methane monooxygenase to increase its catalytic precision with a specific substrate whilst increasing activity by up to 6fold
transcription of sMMO is repressed at Cu2+ concentration above 0.00086 mM per g dry cell weight
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expressed in copper-limited conditions
the enzyme is strongly regulated by the availability of copper. Methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. When Methylosinus trichosporium OB3b is grown in the presence of CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, is very low. Gene mmoX expression increases, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) is added, as does whole-cell sMMO activity, but there is no significant change in the amount of copper associated with Methylosinus trichosporium OB3b. When Methylosinus trichosporium OB3b is grown in the absence of CuCl2, the mmoX expression level is high but decreases by several orders of magnitude when copper prebound to SB2-Mb (Cu-SB2-Mb) is added, and biomass-associated copper is increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb has no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. Gene mbnA expression is reduced when Cu-SB2-Mb is added in both the absence and presence of CuCl2. Methanobactin acts as a general signaling molecule in methanotrophs
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme component MMOB from Escherichia coli is solubilized from the 12000 x g pellet with 8 M urea, followed by dilution to 1 mg/ml protein for enzyme refolding
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
-
high particulate methane monooxygenase activity may contribute to the synthesis of poly-beta-hydroxybutyrate in the cell, which may be used to improve the yield of poly-beta-hydroxybutyrate in methanotrophs. Poly-beta-hydroxybutyrate content of strain OB3b can reach the highest level in the shortest time as compared to other methanotrophs. Nutrients deficiency condition is beneficial for strain IMV3011 to synthesize PHB whereas it is not beneficial for other strains
degradation
-
sMMO can be used for biodegradation of mixtures of chlorinated solvents, i.e., trichloroethylene, trans-dichloroethylene, and vinyl chloride. If the concentrations are increased to 0.1 mM, sMMO-expressing cells grow slower and degrade less of these pollutants in a shorter amount of time than pMMO
environmental protection
A0A2D2D5X0; A0A2D2D0T8; Q53563; A0A2D2D0X7
pulping wastewater still contains massive refractory organics after biotreatment, with high colority, low biodegradability, and lasting biotoxicity. To eliminate refractory organics in pulping wastewater, a methanotrophic co-metabolic system in a gas cycle Sequencing Batch Biofilm Reactor (gcSBBR) seeded by soil at a ventilation opening of coal mine is quickly built on the 92nd day. The removal rate of COD, colority and TOC is 53.28%, 50.59% and 51.60%, respectively. Analysis of 3D-EEM indicates that glycolated protein-like, melanoidin-like or lignocellulose-like, and humic acid-like decrease by 7.85%, 5.02% and 1.74%, respectively
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AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Tonge, G.M.; Harrison, D.E.F.; Higgins, I.J.
Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b
Biochem. J.
161
333-344
1977
Methylosinus trichosporium
Stirling, D.I.; Dalton, H.
Properties of the methane mono-oxygenase from extracts of Methylosinus trichosporium OB3b and evidence for its similarity to the enzyme from Methylococcus capsulatus (Bath)
Eur. J. Biochem.
96
205-212
1979
Methylococcus capsulatus, Methylococcus capsulatus Bath, Methylosinus trichosporium
Fox, B.G.; Froland, W.A.; Jollie, D.R.; Lipscomb, J.D.
Methane monooxygenase from Methylosinus trichosporium OB3b
Methods Enzymol.
188
191-202
1990
Methylosinus trichosporium
Dalton, H.; Smith, D.D.S.; Pilkington, S.J.
Towards a unified mechanism of biological methane oxidation
FEMS Microbiol. Lett.
87
201-208
1990
Methylobacterium sp., Methylococcus capsulatus, Methylococcus capsulatus Bath, Methylosinus trichosporium
-
Fox, B.G.; Liu, Y.; Dege, J.E.; Lipscomb, J.D.
Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction
J. Biol. Chem.
266
540-550
1991
Methylosinus trichosporium
Rataj, M.J.; Kauth, J.E.; Donnelly, M.I.
Oxidation of deuterated compounds by high specific activity methane monooxygenase from Methylosinus trichosporium. Mechanistic implications
J. Biol. Chem.
266
18684-18690
1991
Methylosinus trichosporium
Fox, B.G.; Lipscomb, J.D.
Purification of a high specific activity methane monooxygenase hydroxylase component from a type II methanotroph
Biochem. Biophys. Res. Commun.
154
165-170
1988
Methylosinus trichosporium
Fox, B.G.; Froland, W.A.; Dege, J.E.; Lipscomb, J.D.
Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph
J. Biol. Chem.
264
10023-10033
1989
Methylosinus trichosporium
Lontoh, S.; DiSpirito, A.A.; Semrau, J.D.
Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b
Arch. Microbiol.
171
301-308
1999
Methylosinus trichosporium
-
Jahng, D.; Wood, T.K.
Metal ions and chloramphenicol inhibition of soluble methane monooxygenase from Methylosinus trichosporium OB3b
Appl. Microbiol. Biotechnol.
45
744-749
1996
Methylosinus trichosporium
-
Brazeau, B.J.; Lipscomb, J.D.
Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B
Biochemistry
42
5618-5631
2003
Methylosinus trichosporium
Lee, S.W.; Keeney, D.R.; Lim, D.H.; Dispirito, A.A.; Semrau, J.D.
Mixed pollutant degradation by Methylosinus trichosporium OB3b expressing either soluble or particulate methane monooxygenase: can the tortoise beat the hare?
Appl. Environ. Microbiol.
72
7503-7509
2006
Methylosinus trichosporium
Liu, A.; Jin, Y.; Zhang, J.; Brazeau, B.J.; Lipscomb, J.D.
Substrate radical intermediates in soluble methane monooxygenase
Biochem. Biophys. Res. Commun.
338
254-261
2005
Methylosinus trichosporium
Zhang, J.; Lipscomb, J.D.
Role of the C-terminal region of the B component of Methylosinus trichosporium OB3b methane monooxygenase in the regulation of oxygen activation
Biochemistry
45
1459-1469
2006
Methylosinus trichosporium
Zhang, J.; Wallar, B.J.; Popescu, C.V.; Renner, D.B.; Thomas, D.D.; Lipscomb, J.D.
Methane monooxygenase hydroxylase and B component interactions
Biochemistry
45
2913-2926
2006
Methylosinus trichosporium
Shaofeng, H.; Shuben, L.; Jiayin, X.; Jianzhong, N.; Chungu, X.; Haidong, T.; Wei, T.
Purification and biochemical characterization of soluble methane monooxygenase hydroxylase from Methylosinus trichosporium IMV 3011
Biosci. Biotechnol. Biochem.
71
122-129
2007
Methylosinus trichosporium, Methylosinus trichosporium IMV 3011
Borodina, E.; Nichol, T.; Dumont, M.G.; Smith, T.J.; Murrell, J.C.
Mutagenesis of the "leucine gate" to explore the basis of catalytic versatility in soluble methane monooxygenase
Appl. Environ. Microbiol.
73
6460-6467
2007
Methylosinus trichosporium
Hakemian, A.S.; Kondapalli, K.C.; Telser, J.; Hoffman, B.M.; Stemmler, T.L.; Rosenzweig, A.C.
The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b
Biochemistry
47
6793-6801
2008
Methylosinus trichosporium
Mitic, N.; Schwartz, J.K.; Brazeau, B.J.; Lipscomb, J.D.; Solomon, E.I.
CD and MCD studies of the effects of component B variant binding on the biferrous active site of methane monooxygenase
Biochemistry
47
8386-8397
2008
Methylosinus trichosporium
Zhang, Y.; Xin, J.; Chen, L.; Xia, C.
The methane monooxygenase intrinsic activity of kinds of methanotrophs
Appl. Biochem. Biotechnol.
157
431-441
2009
Methylococcus capsulatus, Methylomonas sp., Methylosinus trichosporium, Methylomonas sp. GYJ3, Methylococcus capsulatus HD6T
Farhan Ul-Haque, M.; Kalidass, B.; Vorobev, A.; Baral, B.S.; DiSpirito, A.A.; Semrau, J.D.
Methanobactin from Methylocystis sp. strain SB2 affects gene expression and methane monooxygenase activity in Methylosinus trichosporium OB3b
Appl. Environ. Microbiol.
81
2466-2473
2015
Methylosinus trichosporium
Lock, M.; Nichol, T.; Murrell, J.C.; Smith, T.J.
Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates
FEMS Microbiol. Lett.
364
fnx137
2017
Methylosinus trichosporium (P27353 and P27355 and P27354 and P27356 and Q53563 and Q53562)
Castillo, R.G.; Banerjee, R.; Allpress, C.J.; Rohde, G.T.; Bill, E.; Que, L.; Lipscomb, J.D.; DeBeer, S.
High-energy-resolution fluorescence-detected X-ray absorption of the Q intermediate of soluble methane monooxygenase
J. Am. Chem. Soc.
139
18024-18033
2017
Methylosinus trichosporium (P27353 and P27355 and P27354 and P27356 and Q53563 and Q53562)
Zhang, T.; Zhou, J.; Wang, X.; Zhang, Y.
Coupled effects of methane monooxygenase and nitrogen source on growth and poly-beta-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b
J. Environ. Sci. (China)
52
49-57
2017
Methylosinus trichosporium (P27353 and P27355 and P27354 and P27356 and Q53563 and Q53562)
Lee, S.J.
Hydroxylation of methane through component interactions in soluble methane monooxygenases
J. Microbiol.
54
277-282
2016
Methylococcus capsulatus (P22869 and P18798 and P11987 and P18797 and P22868 and P22867), Methylosinus trichosporium (P27353 and P27355 and P27354 and P27356 and Q53563 and Q53562), Methylococcus capsulatus Bath. (P22869 and P18798 and P11987 and P18797 and P22868 and P22867)
Zhang, S.; Karthikeyan, R.; Fernando, S.
Low-temperature biological activation of methane structure, function and molecular interactions of soluble and particulate methane monooxygenases
Rev. Environ. Sci. Biotechnol.
16
611-623
2017
Methylococcus capsulatus, Methylococcus capsulatus Bath, Methylosinus trichosporium (P27353 and P27355 and P27354 and P27356 and Q53563 and Q53562)
-
Jones, J.C.; Banerjee, R.; Shi, K.; Aihara, H.; Lipscomb, J.D.
Structural Studies of the Methylosinus trichosporium OB3b soluble methane monooxygenase hydroxylase and regulatory component complex reveal a transient substrate tunnel
Biochemistry
59
2946-2961
2020
Methylosinus trichosporium (A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7), Methylosinus trichosporium
Jones, J.C.; Banerjee, R.; Shi, K.; Semonis, M.M.; Aihara, H.; Pomerantz, W.C.K.; Lipscomb, J.D.
Soluble methane monooxygenase component interactions monitored by 19F NMR
Biochemistry
60
1995-2010
2021
Methylosinus trichosporium (A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7)
Jones, J.C.; Banerjee, R.; Semonis, M.M.; Shi, K.; Aihara, H.; Lipscomb, J.D.
X-ray crystal structures of methane monooxygenase hydroxylase complexes with variants of its regulatory component correlations with altered reaction cycle dynamics
Biochemistry
61
21-33
2022
Methylosinus trichosporium (A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7)
Li, Y.; Wang, Y.; Lin, Z.; Wang, J.; He, Q.; Zhou, J.
A novel methanotrophic co-metabolic system with high soluble methane monooxygenase activity to biodegrade refractory organics in pulping wastewater
Biores. Technol.
256
358-365
2018
Methylosinus trichosporium (A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7)
Srinivas, V.; Banerjee, R.; Lebrette, H.; Jones, J.C.; Aurelius, O.; Kim, I.S.; Pham, C.C.; Gul, S.; Sutherlin, K.D.; Bhowmick, A.; John, J.; Bozkurt, E.; Fransson, T.; Aller, P.; Butryn, A.; Bogacz, I.; Simon, P.; Keable, S.; Britz, A.; Tono, K.; Kim, K.S.; Park, S.Y.; Lee, S.J.; Park, J.; Alonso-Mori, R.; Fu, F.u.l.
High-resolution XFEL structure of the soluble methane monooxygenase hydroxylase complex with its regulatory component at ambient temperature in two oxidation states
J. Am. Chem. Soc.
142
14249-14266
2020
Methylosinus trichosporium (A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7)
Schulz, C.E.; Castillo, R.G.; Pantazis, D.A.; DeBeer, S.; Neese, F.
Structure-spectroscopy correlations for intermediate Q of soluble methane monooxygenase insights from QM/MM calculations
J. Am. Chem. Soc.
143
6560-6577
2021
Methylosinus trichosporium (A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7)
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Transporter Classification Database (TCDB): 9.B.280.1.1
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