Literature summary for 1.14.14.5 extracted from

Liew, J.J.M.; El Saudi, I.M.; Nguyen, S.V.; Wicht, D.K.; Dowling, D.P.
Structures of the alkanesulfonate monooxygenase MsuD provide insight into C-S bond cleavage, substrate scope, and an unexpected role for the tetramer (2021), J. Biol. Chem., 297, 100823 .

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Cloned(Commentary)

Cloned (Comment)	Organism
gene msuD, genetic structure, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Pseudomonas fluorescens

Crystallization (Commentary)

Crystallization (Comment)	Organism
alkanesulfonate monooxygenase unliganded and with a bound flavin and alkanesulfonate, co-crystallization with greater than 5fold molar excess of both ligands, sitting drop vapor diffusion, mixing of 0.001 ml of 8 mg/ml protein solution with 0.001 ml of reservoir solution containing 12-18% w/v PEG 3350, and either 0.14-0.20 M succinate or 0.2-0.3 M sodium acetate set up at room temperature, equilibration againt 0.6 ml of reservoir solution, soaking of crystals in 0.002 ml of reservoir solution supplemented with 2 mM FMN and 2 mM methanesulfonate and 0.001 ml containing MsuD crystals and incubated for over 16 h at 18°C, X-ray diffraction structure determination and analysis at 2.4-2.8 A resolution, ternary-MsuD crystallized in the space group P61 with four MsuD chains per asymmetric unit, arranged as a dimer-of-dimers. In the absence of ligands, MsuD crystallizes in space group P21 with two MsuD tetramers (chains A/B/C/D and E/F/G/H) per asymmetric unit. The active site of MsuD is solvent exposed without FMN bound. Molecular replacement of the unliganded MsuD dataset using the structure of homologue SsuD from Escherichia coli strain K12 (PDB ID 1NQK) as template, modeling	Pseudomonas fluorescens
structures of MsuD in different liganded states. The active site of MsuD is solvent exposed without FMN bound. Substrate methanesulfonate is positioned closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. The protein C terminus functions in stabilizing tetramer formation and the alkanesulfonate-binding site. MsuD is a small- to medium-chain alkanesulfonate monooxygenase	Pseudomonas fluorescens

Crystallization (Comment)

Organism

alkanesulfonate monooxygenase unliganded and with a bound flavin and alkanesulfonate, co-crystallization with greater than 5fold molar excess of both ligands, sitting drop vapor diffusion, mixing of 0.001 ml of 8 mg/ml protein solution with 0.001 ml of reservoir solution containing 12-18% w/v PEG 3350, and either 0.14-0.20 M succinate or 0.2-0.3 M sodium acetate set up at room temperature, equilibration againt 0.6 ml of reservoir solution, soaking of crystals in 0.002 ml of reservoir solution supplemented with 2 mM FMN and 2 mM methanesulfonate and 0.001 ml containing MsuD crystals and incubated for over 16 h at 18°C, X-ray diffraction structure determination and analysis at 2.4-2.8 A resolution, ternary-MsuD crystallized in the space group P61 with four MsuD chains per asymmetric unit, arranged as a dimer-of-dimers. In the absence of ligands, MsuD crystallizes in space group P21 with two MsuD tetramers (chains A/B/C/D and E/F/G/H) per asymmetric unit. The active site of MsuD is solvent exposed without FMN bound. Molecular replacement of the unliganded MsuD dataset using the structure of homologue SsuD from Escherichia coli strain K12 (PDB ID 1NQK) as template, modeling

Pseudomonas fluorescens

structures of MsuD in different liganded states. The active site of MsuD is solvent exposed without FMN bound. Substrate methanesulfonate is positioned closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. The protein C terminus functions in stabilizing tetramer formation and the alkanesulfonate-binding site. MsuD is a small- to medium-chain alkanesulfonate monooxygenase

Pseudomonas fluorescens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
an alkanesulfonate + FMNH2 + O2	Pseudomonas fluorescens	-	an aldehyde + FMN + sulfite + H2O	-	?
an alkanesulfonate + FMNH2 + O2	Pseudomonas fluorescens Pf0-1	-	an aldehyde + FMN + sulfite + H2O	-	?
methanesulfonate + FMNH2 + O2	Pseudomonas fluorescens	-	formaldehyde + FMN + sulfite + H2O	-	?
methanesulfonate + FMNH2 + O2	Pseudomonas fluorescens Pf0-1	-	formaldehyde + FMN + sulfite + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas fluorescens	Q3K9A1	-	-
Pseudomonas fluorescens Pf0-1	Q3K9A1	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration	Pseudomonas fluorescens

Reaction

Reaction	Comment	Organism	Reaction ID
an alkanesulfonate + FMNH2 + O2 = an aldehyde + FMN + sulfite + H2O	proposed mechanism for two-component flavin-dependent alkanesulfonate monooxygenases based on the ternary-MsuD structure, overview	Pseudomonas fluorescens	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate + FMNH2 + O2	HEPES	Pseudomonas fluorescens	? + FMN + sulfite + H2O	-	?
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate + FMNH2 + O2	HEPES	Pseudomonas fluorescens Pf0-1	? + FMN + sulfite + H2O	-	?
3-(N-morpholino)propanesulfonate + FMNH2 + O2	MOPS	Pseudomonas fluorescens	? + FMN + sulfite + H2O	-	?
an alkanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens	an aldehyde + FMN + sulfite + H2O	-	?
an alkanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens Pf0-1	an aldehyde + FMN + sulfite + H2O	-	?
methanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens	formaldehyde + FMN + sulfite + H2O	-	?
methanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens Pf0-1	formaldehyde + FMN + sulfite + H2O	-	?
additional information	when both the lid and C-terminus are ordered and bound in ternary-MsuD, the active site appears completely enclosed from bulk solvent. The apparent volume is larger than methanesulfonate (MS-), consistent with previously observed activity against larger sulfonate substrates. Therefore, molecular docking of substrates ranging in size from pentanesulfonate to PIPES is explored. Docking returns possible poses with the sulfonate moiety in a similar orientation as observed for MS-, but with variable positioning of alkyl groups. Molecular docking defines MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Substrate binding structures for methanesulfonate (alkanesulfonate), FMNH2, and O2, involving the enzyme's C-terminuns, overview	Pseudomonas fluorescens	?	-	-
additional information	when both the lid and C-terminus are ordered and bound in ternary-MsuD, the active site appears completely enclosed from bulk solvent. The apparent volume is larger than methanesulfonate (MS-), consistent with previously observed activity against larger sulfonate substrates. Therefore, molecular docking of substrates ranging in size from pentanesulfonate to PIPES is explored. Docking returns possible poses with the sulfonate moiety in a similar orientation as observed for MS-, but with variable positioning of alkyl groups. Molecular docking defines MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Substrate binding structures for methanesulfonate (alkanesulfonate), FMNH2, and O2, involving the enzyme's C-terminuns, overview	Pseudomonas fluorescens Pf0-1	?	-	-
octanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens	octanal + FMN + sulfite + H2O	-	?
octanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens Pf0-1	octanal + FMN + sulfite + H2O	-	?
octanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens	octaldehyde + FMN + sulfite + H2O	-	?
pentanesulfonate + FMNH2 + O2	-	Pseudomonas fluorescens	pentaldehyde + FMN + sulfite + H2O	-	?
pentanesulfonate + FMNH2 + O2	62% of the activtiy with octanesulfonate	Pseudomonas fluorescens	pentaldehyde + FMN + sulfite + H2O	-	?
piperazine-N,N'-bis(2-ethanesulfonate) + FMNH2 + O2	PIPES	Pseudomonas fluorescens	? + FMN + sulfite + H2O	-	?

Subunits

Subunits	Comment	Organism
More	MsuD adopts a classic (beta/alpha)8 TIM barrel fold with four insertion segments (ISs) to the (beta/alpha)8 core of MsuD: IS-1 includes two beta-strands (labeled beta1a/b), IS-2 includes an alpha-helix (labeled alpha4a), IS-3 includes a beta-hairpin (labeled beta4a/b); and IS-4 includes three alpha-helices (labeled alpha7a/b/c), a 310 helix (labeled nu7d), and three short beta-strands (labeled beta7a/b/c). In addition, MsuD contains an extended protein C-terminus involved in catalysis	Pseudomonas fluorescens
oligomer	the ligand free enzyme forms a dimer-of-dimers structure in cyrstals, while the liganded enzyme froms a dimer-of-tetramers, overview	Pseudomonas fluorescens

Synonyms

Synonyms	Comment	Organism
alkanesulfonate monooxygenase	-	Pseudomonas fluorescens
msuD	-	Pseudomonas fluorescens
Pfl01_3916	-	Pseudomonas fluorescens
SsuD	-	Pseudomonas fluorescens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Pseudomonas fluorescens

Cofactor

Cofactor	Comment	Organism	Structure
FMNH2	dependent on, not permanently enzyme bound, binding structure, overview	Pseudomonas fluorescens

General Information

General Information	Comment	Organism
evolution	the enzymes SfnG, MsuC, and MsuD are members of a small subset of flavin-dependent monooxygenases that are characterized by their use of reduced flavin as a cosubstrate rather than a cofactor. Termed two-component flavin-dependent monooxygenases, members of this family lack an NAD(P)H-binding site and therefore require a separate reduced NAD(P)H:oxidized flavin mononucleotide (FMN) oxidoreductase to provide the FMNH- cosubstrate	Pseudomonas fluorescens
metabolism	small- to medium-chain alkanesulfonate monooxygenase enzyme MsuD plays a role in the sulfur assimilation pathway. The flavin-dependent monooxygenases SfnG, MsuC, and MsuD convert DMSO2 to sulfite. SfnG converts DMSO2 to methanesulfinate (MSI-), MsuC oxidizes MSI- to methanesulfonate (MS-), and MsuD catalyzes the conversion of MS- to sulfite. Together SfnG and MsuD are responsible for sequential cleavage of the two C-S bonds of DMSO2, and each methyl group is presumed to be oxidized to formaldehyde	Pseudomonas fluorescens
additional information	molecular docking, structure-function analysis, roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding, overview. These structures position MS- closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C-terminus at the tetramer interface identifying a function of the protein C-terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. The structures of MsuD with and without ligands support ordered binding for FMNH- and MS-, and the preferential binding of FMN first within chains A/C and E/G is suggestive of possible cooperativity. Without ligands, the active site lid, the sulfonate-binding loop, and the protein C terminus are disordered	Pseudomonas fluorescens
physiological function	bacterial two-component flavin-dependent monooxygenases cleave the stable C-S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS-) to sulfite, completing the sulfur assimilation process during sulfate starvation	Pseudomonas fluorescens