Information on EC 1.1.1.132 - GDP-mannose 6-dehydrogenase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY
1.1.1.132
-
RECOMMENDED NAME
GeneOntology No.
GDP-mannose 6-dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
GDP-D-mannose + 2 NAD+ + H2O = GDP-D-mannuronate + 2 NADH + 2 H+
show the reaction diagram
class of 4-electron transfer dehydrogenases
-
GDP-D-mannose + 2 NAD+ + H2O = GDP-D-mannuronate + 2 NADH + 2 H+
show the reaction diagram
also acts on the corresponding deoxynucleoside diphosphate derivative as a substrate
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
alginate biosynthesis I
-
alginate biosynthesis II
-
Amino sugar and nucleotide sugar metabolism
-
Fructose and mannose metabolism
-
SYSTEMATIC NAME
IUBMB Comments
GDP-D-mannose:NAD+ 6-oxidoreductase
Also acts on the corresponding deoxynucleoside diphosphate derivative as a substrate.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
algD
-
gene name
GDP mannose dehydrogenase
-
-
-
-
GDP-mannose dehydrogenase
-
-
GDP-mannose dehydrogenase
-
-
GDP-mannose dehydrogenase
P11759
-
GDP-mannose dehydrogenase
A4ZZ81
-
GMD
-
-
-
-
guanosine diphospho-D-mannose dehydrogenase
-
-
-
-
guanosine diphospho-D-mannose dehydrogenase
-, P11759
-
guanosine diphosphomannose dehydrogenase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
37250-63-8
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
NRRL B1973
-
-
Manually annotated by BRENDA team
Arthrobacter sp. NRRL B1973
NRRL B1973
-
-
Manually annotated by BRENDA team
NCIB 8789, water soluble polysaccharide producing strains E, W and O
-
-
Manually annotated by BRENDA team
SM52B, mutant of A. vinelandii NCIB 9068, alginate-prducing ability, no activity with sorbitol as sole carbon source during batch growth, enzyme not produced, loss of ability to synthesise alginate during prolonged continuous culture after 300 h for mutant and more than 1000 h for parent strain, ability transferable by conjugation
-
-
Manually annotated by BRENDA team
; enzyme overproduced using a plasmid vector containing algD under control of the tac promoter; mucoid strain 8821
-
-
Manually annotated by BRENDA team
A22 alg+, selected from mucoid strain IPA22
-
-
Manually annotated by BRENDA team
algD gene overproduced in strain 8822 using a plasmid vector containing algD under control of the tac promoter
-
-
Manually annotated by BRENDA team
enzyme overproduced using 3 different plasmid vectors containing algD under control of the tac promoter expressed in non-mucoid strain PAO1 and mucoid strain PAO568, pPT3 and pPT4 are forward and reverse clones contain leader sequence with possible transcriptional/translational regulatory sequences of algD gene, no significant activity with pMMB66EH and pPT4 in either IPTG induced or non induced Escherichia coli, mucoid and non-mucoid Pseudomonas aeruginosa strains
-
-
Manually annotated by BRENDA team
expressed in several mutants; no activity in nonmucoid mutants
-
-
Manually annotated by BRENDA team
mucoid clinical isolate PT-1578 and non-mucoid PAO1, very low activity of PAO1 regardless whether using nutrient-rich or poor medium, low activity with PT-1578 on enriched, increased on nutritionally poor medium
-
-
Manually annotated by BRENDA team
mucoid strain PA7563; no activity in nonmucoid mutants
-
-
Manually annotated by BRENDA team
mucoid strains of cystic fibrosis patients 8821 and its high-alginate-producing spontaneously variant 8821M, enzyme overproduced using 2 different plasmid vectors containing algD under control of the tac promoter mobilized into both strains
-
-
Manually annotated by BRENDA team
mucoid strains of cystic fibrosis patients 8821, its high-alginate-producing spontaneously variant 8821M, IST23, IST27 and IST30, mucoid strains derived from the spontaneous nonmucoid strains during prolonged incubation under nutrient limitation in acetamide broth (Nm), rich in PolyP, no activity in spontaneous swiched nonmucoid variants (N) of initial isolated mucoid strains
-
-
Manually annotated by BRENDA team
overexpression of tac promoter controlled algD gene containing different plasmids in mucoid strains FRD1 and PAO568 and non-mucoid strains FRD2 and PAO1
-
-
Manually annotated by BRENDA team
-
A4ZZ81
UniProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
deoxy-GDP-D-mannose + NAD+ + H2O
deoxy-GDP-D-mannuronate + NADH
show the reaction diagram
-
-
-
-
?
deoxy-GDP-D-mannose + NAD+ + H2O
deoxy-GDP-D-mannuronate + NADH
show the reaction diagram
-
-
-
?
deoxy-GDP-D-mannose + NAD+ + H2O
deoxy-GDP-D-mannuronate + NADH
show the reaction diagram
Arthrobacter sp. NRRL B1973
-
-
-
-
?
GDP-D-mannose + NAD+ + H2O
GDP-D-mannuronate + NADH
show the reaction diagram
-
-
-
-
?
GDP-D-mannose + NAD+ + H2O
GDP-D-mannuronate + NADH
show the reaction diagram
-
-
-
-
?
GDP-D-mannose + NAD+ + H2O
GDP-D-mannunorate + NADH
show the reaction diagram
-
-, the enzyme catalyzes the first committed step in alginate biosynthesis
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
ir
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
ir
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
ir
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
ir
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
-
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
?
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
-
ir
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
-
-
water-soluble and insoluble polysaccharides are produced, containing glucuronic and mannuronic acid, glucose and rhamnose, GDP-D-mannose dehydrogenase activity detected in 30 h cultures and 36 h cultures containing only water-insoluble capsular or water-soluble polysaccharides, respectively
-
GDP-mannose + NAD+ + H2O
GDP-mannuronate + NADH
show the reaction diagram
Arthrobacter sp. NRRL B1973
-
-
-
?
additional information
?
-
-
key enzyme in alginate biosynthesis, no substrate: D-mannose, UDP-D-mannose, UDP-D-glucose, TDP-D-glucose, CDP-D-glucose
-
-
-
additional information
?
-
-
GMD is the key enzyme in alginate biosynthesis. Biofilm-forming bacteria Pseudomonas aeruginosa is a common pathogen in mechanically ventilated newborns, which can cause life-threatening infections
-
-
-
additional information
?
-
A4ZZ81
algD is localized in an alginate biosynthesis cluster
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
GDP-D-mannose + NAD+ + H2O
GDP-D-mannunorate + NADH
show the reaction diagram
-
the enzyme catalyzes the first committed step in alginate biosynthesis
-
-
?
additional information
?
-
-
key enzyme in alginate biosynthesis
-
-
-
additional information
?
-
-
GMD is the key enzyme in alginate biosynthesis. Biofilm-forming bacteria Pseudomonas aeruginosa is a common pathogen in mechanically ventilated newborns, which can cause life-threatening infections
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
NAD+
-
cooperative behaviour with respect to NAD+ binding, sensitive to pH
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
CoCl2
-
at 1 mM CoCl2 44% enzymatic activity
CuSO4
-
at 1 mM CuSO4 no enzymatic activity
FeCl3
-
at 1 mM FeCl3 49% enzymatic activity
FeSO4
-
at 1 mM FeSO4 77% enzymatic activity
K+
-
increases activity
Na+
-
increases activity
PCMB
-
0.025 mM, 0.8% activity
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
5'-(3-hydroxypropyl)-amino-5'-deoxy-guanosine
-
only after preincubation with enzyme
5'-azido-5'-deoxy-guanosine
-
only after preincubation with enzyme
5'-azido-5'-deoxy-N2-2',3'-O-triacetylguanosine
-
only after preincubation with enzyme
5'[[[[(2'',3'',4'',6''-tetra-O-acetyl 6''-ethynyl)alpha-D-mannopyranosyl oxy]-carbonyl]amino]sulfonyl]-2',3'-isopropylidene N2-acetyl guanosine
-
GDP-analog, 0.1 mM, 60% inhibition
ambroxol
-
i.e. 2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine, inhibits GMD activity, reduces the production of alginate, the expression of the important genes, and affects the structure of mucoid Pseudomonas aeruginosa biofilms, overview; reduces activity of the key enzyme GMD in alginate biosynthesis in a concentration-dependent manner
ATP
-
; 26% inhibition at 1 mM
D-maltose
-
17% inhibition at 1 mM
erythromycin
-
potent dose-dependent inhibitory effect, inhibition of biofilm formation, PT-1578
GDP-D-glucose
-
; 25% inhibition at 1 mM
GMP
-
competitive, 52% inhibition at 1 mM; competitive, most potent
guanosine
-
slight inhibition only after preincubation with enzyme
iodacetamide
-
40% inactivation within 30 min, complete reactivation by dithiothreitol
iodoacetamide
-
40% inactivation after 30 min preincubation
N2-acetyl-2',3'-isopropylidene guanosine
-
only after preincubation with enzyme
N2-acetyl-5'-amino-5'-deoxy-2',3'-O-acetylguanosine
-
only after preincubation with enzyme
N2-acetyl-5'-amino-5'-deoxy-guanosine
-
only after preincubation with enzyme
N2-acetyl-guanosine
-
only after preincubation with enzyme
NADPH
-
weak inhibitor of reaction
p-hydroxymercuribenzoate
-
96% inactivation after 5 min preincubation; 96% inactivation within 5 min, complete reactivation by dithiothreitol
penicillic acid
-
irreversible inactivation
tetra-O-acetylmannopyranose
-
GDP-analog, 0.1 mM, 75% inhibition
-
midecamycin
-
weaker inhibition, no biofilm destruction, PT-1578
additional information
-
full reactivation by dithiothreitol (25 mM) after p-hydroxymercuribenzoate or iodoacetamide inactivation
-
additional information
-
strains which resist 0.005 mg/ml of tobramycin treated by 5'-(3-hydroxypropyl)-amino-5'-deoxy-guanosine, tetra-O-acetylmannopyranose and 5'[[[[(2'',3'',4'',6''-tetra-O-acetyl 6''-ethynyl)alpha-D-mannopyranosyl oxy]-carbonyl]amino]sulfonyl]-2',3'-isopropylidene N2-acetyl guanosine recover 20, 75 and 85% of their sensisivity towards tobramycin
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.084
-
deoxy-GDP-mannose
-
-
0.009
-
GDP-D-mannose
-
pH 8.0, 5C, recombinant wild-type enzyme
0.0149
-
GDP-D-mannose
-
; 24C, pH 8.0
0.023
-
GDP-D-mannose
-
pH 8.0, 5C, recombinant mutant C213A
0.045
-
GDP-D-mannose
-
pH 8.0, 5C, recombinant mutant C268A
0.095
-
GDP-D-mannose
-
pH and temperature not specified in the publication
0.086
-
NAD+
-
pH and temperature not specified in the publication
0.1
-
NAD+
-
pH 8.0, phosphate buffer
0.185
-
NAD+
-
24C, pH 8.0; cosubstrate GDP-D-mannose
0.19
-
NAD+
-
pH 8.0, 5C, recombinant mutant C213A
0.21
-
NAD+
-
pH 8.0, 5C, recombinant wild-type enzyme
0.26
-
NAD+
-
cosubstrate 0.3 mM GDP-mannose
0.51
-
NAD+
-
pH 8.0, 5C, recombinant mutant C268A
0.017
-
GDP-mannose
-
-
additional information
-
additional information
-
in phosphate buffer, enzyme shows Michaelis-Menten kinetics, in Tris buffer, enzyme shows marked cooperativity with respect to NAD+ binding. Phosphate and GMP are allosteric effectors
-
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0227
-
GMP
-
24C, pH 8.0
additional information
-
additional information
-
inactivation kinetics of recombinant wild-type and mutant enzymes with penicillic acid
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.00015
-
-
+/-0.00001, S17-1/pPT4, with IPTG
0.00017
-
-
+/-0.00002, S17-1/pMMB66EH and S17-1/pPT4 without IPTG
0.00022
-
-
+/-0.00001, S17-1/pMMB66EH, with IPTG
0.00034
-
-
+/-0.00006, S17-1/pPT3, without IPTG
0.0004
-
-
+/-0.00008, PAO1/pMMB66EH, mid-exponential, without IPTG
0.00056
-
-
+/-0.00024, PAO1/pMMB66EH, early-stationary, with IPTG
0.00076
-
-
+/-0.00016, FRD2/pMMB66EH, mid-exponential, with IPTG
0.00089
-
-
+/-0.0002, FRD2/pMMB66EH, mid-exponential, without IPTG
0.0009
-
-
+/-0.0002, PAO1/pMMB66EH, with IPTG; +/-0.0003, PAO1/pMMB66EH, without IPTG
0.00091
-
-
+/-0.00042, PAO1/pMMB66EH, mid-exponential, with IPTG
0.00094
-
-
+/-0.00052, FRD2/pMMB66EH, early-stationary, with IPTG
0.00106
-
-
+/-0.00062, PAO1/pMMB66EH, early-stationary, without IPTG
0.0013
-
-
+/-0.00004, FRD2/pMMB66EH, early-stationary, without IPTG
0.002
0.015
-
mucoid forms, activity correlated with alginate production and level of transcription of algD gene
0.002
-
-
inorganic phosphate limitation, 85 h after changing carbon source from sorbitol to sucrose
0.0025
-
-
oxygen limitation
0.0058
-
-
+/-0.002, 8821/pMMB24, not IPTG induced
0.00685
-
-
+/-0.00228, FRD1/pMMB66EH, mid-exponential, with IPTG
0.00749
-
-
+/-0.00187, PAO1/pPT3, mid-exponential, without IPTG
0.00936
-
-
+/-0.00063, PAO1/pPT3, early-stationary, without IPTG
0.00968
-
-
+/-0.00155, FRD1/pMMB66EH, mid-exponential, without IPTG
0.00992
-
-
+/-0.00105, PAO568/pMMB66EH, mid-exponential, without IPTG
0.01097
-
-
+/-0.00195, FRD2/pPT4, early-stationary, with IPTG
0.01232
-
-
+/-0.00397, PAO568/pMMB66EH, mid-exponential, with IPTG
0.01249
-
-
+/-0.00113, FRD2/pPT4, early-stationary, without IPTG
0.0128
-
-
+/-0.0018, 8821/pVD211, not IPTG induced
0.01283
-
-
+/-0.00348, FRD2/pPT3, early-stationary, without IPTG
0.013
-
-
+/-0.003, PAO1/pPT3, without IPTG
0.01312
-
-
+/-0.0031, FRD2/pPT3, mid-exponential, without IPTG
0.014
-
-
+/-0.0014, PAO1/pPT4, with IPTG
0.01401
-
-
+/-0.00147, PAO1/pPT4, early-stationary, with IPTG
0.01436
-
-
+/-0.00303, FRD1/pPT3, mid-exponential, without IPTG
0.0147
-
-
+/-0.0014, PAO1/pPT4, without IPTG
0.01472
-
-
+/-0.00135, PAO1/pPT4, early-stationary, without IPTG
0.0176
-
-
+/-0.0009, PAO568/pMMB66EH, with IPTG
0.0182
-
-
+/-0.0003, PAO568/pMMB66EH, without IPTG
0.01886
-
-
+/-0.00526, PAO568/pMMB66EH, early-stationary, without IPTG
0.01891
-
-
+/-0.0001, PAO568/pPT3, mid-exponential, without IPTG
0.0223
-
-
+/-0.0011, PAO568/pPT3, without IPTG
0.0226
-
-
+/-0.0025, 8821/pVD211, induced by 0.5 mM IPTG
0.02322
-
-
+/-0.00432, PAO568/pMMB66EH, early-stationary, with IPTG
0.02418
-
-
+/-0.00628, FRD1/pMMB66EH, early-stationary, without IPTG
0.0251
-
-
+/-0.0027, PAO568/pPT4, without IPTG
0.02514
-
-
+/-0.00274, PAO568/pPT4, early-stationary, without IPTG
0.0254
-
-
+/-0.004, PAO568/pPT4, with IPTG
0.02542
-
-
+/-0.00139, PAO568/pPT4, early-stationary, with IPTG
0.02574
-
-
+/-0.00581, FRD1/pMMB66EH, early-stationary, with IPTG
0.0265
-
-
+/-0.003, 8821/pVD211, induced by 3 mM IPTG
0.02671
-
-
+/-0.00459, FRD1/pPT4, early-stationary, with IPTG
0.028
-
-
+/-0.0029, 8821M/pMMB24, not IPTG induced
0.02905
-
-
+/-0.00427, FRD1/pPT4, early-stationary, without IPTG
0.02994
-
-
+/-0.0066, PAO568/pPT3, early-stationary, without IPTG
0.03208
-
-
+/-0.00629, FRD1/pPT3, early-stationary, without IPTG
0.0579
-
-
+/-0.0029, 8821/pVD211, not IPTG induced
0.084
-
-
+/-0.0019, 8821/pVD211, induced by 0.5 mM IPTG
0.0942
-
-
+/-0.004, 8821/pVD211, induced by 3 mM IPTG
0.1259
-
-
+/-0.02878, PAO1/pPT3, mid-exponential, with IPTG
0.1364
-
-
+/-0.01055, FRD2/pPT3, mid-exponential, with IPTG
0.1627
-
-
+/-0.03188, PAO568/pPT3, mid-exponential, with IPTG
0.1758
-
-
+/-0.03971, FRD1/pPT3, mid-exponential, with IPTG
0.1895
-
-
+/-0.00702, S17-1/pPT3, with IPTG
0.1919
-
-
+/-0.00268, PAO568/pPT3, early-stationary, with IPTG
0.2037
-
-
+/-0.06998, PAO1/pPT3, early-stationary, with IPTG
0.2041
-
-
+/-0.00932, PAO1/pPT3, with IPTG
0.2097
-
-
+/-0.03274, FRD1/pPT3, early-stationary, with IPTG
0.2196
-
-
+/-0.05517, FRD2/pPT3, early-stationary, with IPTG
0.2378
-
-
+/-0.0552, PAO568/pPT3, with IPTG
0.245
-
-
-
1068
-
-
24C, pH 8.0
additional information
-
-
increase of activity has only limited effect on the stimulation of alginate synthesis, existance of other rate-limiting enzymatic step suggested
additional information
-
-
presence of 1 mM GDP-mannose, GDP-glucose, GMP, GDP, GTP or cGMP retards tryptic digest of enzyme and protects subsequent loss of enzyme activity, amino-terminal domain contains substrate and cofactor binding sites, carboxy-terminal domain is essential for catalytic activity
additional information
-
-
higher enzyme activities after IPTG induction only for pPT3 containing strains, low GDP-mannuronate production with pMMB66EH only detectable in mucoid strains, higher with pPT3 with increase in cells of mucoid and non-mucoid strains with IPTG, alginate formation occurs only in mucoid strains without significant increase with IPTG, later enzyme of alginate biosynthesis suggested to become rate-limiting
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8.2
-
-
Tris buffer
8.75
-
-
assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8.75
9
-
optimal pH range, enzyme inactive below pH 7
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
37
-
-
assay at
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
34000
-
-
SDS-PAGE
47600
-
-
estimation of open reading frame
48000
-
-
SDS-PAGE, Escherichia coli and Pseudomonas aeruginosa expressed polypeptide
48000
-
-
SDS-PAGE
70000
-
-
gel filtration
290000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
dimer
-
2 * 35000, gel filtration
hexamer
-
6 * 48000, SDS-PAGE
additional information
-
N-terminal amino acid sequence
additional information
-
forms a domain-swapped dimer with two polypeptide chains contributing to each active site, crystallization data
additional information
-
peptide mapping of recombinant wild-type and mutant enzymes with MALDI-TOF mass spectrometry
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ternary complex with NAD and product GDP-mannuronic acid
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
30
-
-
enzyme partly inactivated above 30C
45
-
-
enzyme fully inactivated at 45C after 15 min
50
-
-
above, 2-3 min, loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
repeated freezing/thawing leads to decrease of activity
-
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-15C, protein concentration above 1 mg/ml, at least 1 month
-
-70C, 1-3 mg/ml purified protein, 50 mM triethanolamine acetate, pH 7.6, 1 mM DTT
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ammonium sulfate fractionation, affinity chromatography
-
protamine sulfate fractionation
-
using Ni-NTA chromatography
-
ammonium sulfate precipitation
-
pH/heat treatment acetone precipitation HPLC gel filtration chromatography
-
protamine sulfate precipitation, gel exclusion, ion exchange chromatography
-
recombinant wild-type and mutant enzymes from Escherichia coli by gel filtration of enzyme with tightly bound GDP-mannuronic acid and NAD+, dialysis and ion exchange chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in Escherichia coli as a His-tagged fusion protein
-
gene algD, overexpression of wild-type and mutant enzymes in Escherichia coli
-
algD cloned into a pMD18 T-vector and transformed into Escherichia coli DH5alpha-competent cells
A4ZZ81
EXPRESSION
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
in Azotobacter vinelandii GacA regulates alginate synthesis through its control of rsmZ1 and rsmZ2 transcription, which in turn relieves the repressing activity of RsmA on algD expression
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
C213A
-
site-directed mutagenesis, 1.8fold increased Vmax compared to the wild-type enzyme
C268A
-
site-directed mutagenesis, 250fold reduced Vmax, 5fold increased KM, and reduced sensitivity to penicillic acid inactivation compared to the wild-type enzyme
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
inactivation by iodacetamide or p-hydroxymercuribenzoate is completely reversible
-