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Literature summary extracted from

  • Yoshiwara, K.; Watanabe, S.; Watanabe, Y.
    Crystal structure of L-rhamnose 1-dehydrogenase involved in the nonphosphorylative pathway of L-rhamnose metabolism in bacteria (2021), FEBS Lett., 595, 637-646 .
    View publication on PubMed

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.378 purified recombinant enzyme in ligand-free, NAD+-bound, NADP+-bound, and L-rhamnose- and NAD+-bound forms, sitting drop vapor diffusion method, mixing of 500 nl of 10 mg/ml protein in 20 mM Tris/HCl, pH 8.0, and 150 mM NaCl, with 500 nl of reservoir olution containing 100 mM Tris/HCl, pH 8.5, 200 mM Li2SO4, and 25% w/v PEG 3350, with or without coenzyme, for the apoenzyme and for the NADP+-bound form,, and 100 mM Tris/HCl, pH 8.0, 200 mM NaCl, 24.5% w/v PEG 3350, and 3% v/v glycerol only for the NAD+-/L-rhamnose-bound form, equilibration against 0.07 ml of reservoir solution, at 20°C, X-ray diffraction structure determination and analysis at 1.9, 2.1, 2.4, and 1.6 A resolution, respectively, molecular replacement using the structure of 3-oxoacyl-(acyl carrier protein) reductase from Bacillus anthracis (PDB ID 2UVD) as the search model, modeling Azotobacter vinelandii

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.378 D200A site-directed mutagenesis, the mutant shows 16% activity with L-rhamnose compared to wild-type Azotobacter vinelandii
1.1.1.378 D200H site-directed mutagenesis, the mutant shows 22% activity with L-rhamnose compared to wild-type Azotobacter vinelandii
1.1.1.378 F99A site-directed mutagenesis, the mutant is inactive Azotobacter vinelandii
1.1.1.378 F99Y site-directed mutagenesis, the mutant is inactive Azotobacter vinelandii
1.1.1.378 I196A site-directed mutagenesis, the mutant is inactive Azotobacter vinelandii
1.1.1.378 Q156A site-directed mutagenesis, the mutant is almost inactive Azotobacter vinelandii
1.1.1.378 R15T site-directed mutagenesis, the mutant of AvRhaDH shows a Km value for NADP+ decreased by 116fold, which increases the ratio of NADP+ to NAD+ (0.188) 157fold, from that of the wild-type enzyme (18.5) Azotobacter vinelandii
1.1.1.378 S37H site-directed mutagenesis, the mutant shows an altered ratio of NADP+ to NAD+ (0.101) compared to that of the wild-type enzyme (18.5) Azotobacter vinelandii
1.1.1.378 T191F site-directed mutagenesis, the mutant shows 3.9% activity with L-rhamnose compared to wild-type Azotobacter vinelandii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.378 0.194
-
 NADP+ recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 1.5 2 NAD+ recombinant mutant S37H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 1.81
-
 NAD+ recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 2.23
-
 L-rhamnose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 4.82
-
 L-Lyxose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 5.52
-
 NAD+ recombinant mutant R15T, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 7.73
-
 L-rhamnose recombinant mutant D200H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 11.3
-
 L-rhamnose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 12.9
-
 NADP+ recombinant mutant S37H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 13.7
-
 L-rhamnose recombinant mutant D200A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 22.5
-
 NADP+ recombinant mutant R15T, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 22.6
-
 L-Lyxose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 26.1
-
 L-rhamnose recombinant mutant T191F, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 29.7
-
 L-Lyxose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 37.9
-
 L-rhamnose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 66.2
-
 L-rhamnose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 68.6
-
 L-Mannose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 78.9
-
 L-rhamnose recombinant mutant Q156A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 129
-
 L-Mannose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 148
-
 L-Mannose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 159
-
 L-Mannose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 244
-
 L-Lyxose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.378 L-rhamnose + NADP+ Azotobacter vinelandii
-
 L-rhamnono-1,4-lactone + NADPH + H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.378 Azotobacter vinelandii
-
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.378 L-rhamnose + NAD(P)+ = L-rhamnono-1,4-lactone + NAD(P)H + H+ in putative catalytic mechanism, the C1-OH group of L-rhamnose is deprotonated by Tyr159 as a general basic catalyst, with concurrent transfer of the hydride ion to NAD(P)+, and the pKa value of its hydroxyl group is lowered by Lysl63, leading to the stabilization of the tyrosinate anion at physiological pH Azotobacter vinelandii
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.378 L-lyxose + NAD(P)+
-
 Azotobacter vinelandii ? + NAD(P)H + H+
-
 ?
1.1.1.378 L-mannose + NAD(P)+
-
 Azotobacter vinelandii ? + NAD(P)H + H+
-
 ?
1.1.1.378 L-rhamnose + NAD+ L-rhamnose is the best substrate, preference for NADP+ over NAD+ Azotobacter vinelandii L-rhamnono-1,4-lactone + NADH + H+
-
 ?
1.1.1.378 L-rhamnose + NADP+
-
 Azotobacter vinelandii L-rhamnono-1,4-lactone + NADPH + H+
-
 ?
1.1.1.378 L-rhamnose + NADP+ L-rhamnose is the best substrate, preference for NADP+ over NAD+ Azotobacter vinelandii L-rhamnono-1,4-lactone + NADPH + H+
-
 ?
1.1.1.378 additional information the C5-OH and C6-methyl groups of L-rhamnose are recognized by specific residues of RhaDH through hydrogen bonds and hydrophobic contact, respectively, which contribute to the different substrate specificities from other aldose 1-dehydrogenases in the short-chain dehydrogenase/reductase superfamily Azotobacter vinelandii ?
-
-

Subunits

EC Number Subunits Comment Organism
1.1.1.378 More the subunit of AvRhaDH was a single-domain protein with an alpha/beta doubly wound structure. A seven-stranded parallel beta-sheet (beta3-beta2-beta1-beta4-beta5-beta6-beta7) in the center of the molecule is sandwiched by two arrays of parallel alpha-helices (alpha1 to alpha6), which is a typical dinucleotide binding the Rossmann fold motif including the characteristic sequence Gly-X3-Gly-X-Gly; Gly12-Ala13-Ser14-Arg15-Gly16-Ile17-Gly18. Furthermore, a motif corresponding to Asn92-Asn93-Ala94-Gly95 is previously shown to be important for stabilizing this central beta-sheet in other SDR superfamily enzymes. A small domain containing the alpha7 and two 310 helixes is slightly separated from the main body of the subunit, and contained the flexible region Azotobacter vinelandii

Synonyms

EC Number Synonyms Comment Organism
1.1.1.378 AvRhaDH
-
 Azotobacter vinelandii
1.1.1.378 L-rhamnose 1-dehydrogenase
-
 Azotobacter vinelandii
1.1.1.378 RhaDH
-
 Azotobacter vinelandii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.378 30
-
 oxidation of L-rhamnose Azotobacter vinelandii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.378 5.77
-
 L-Mannose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 9.55
-
 L-Mannose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 11.35
-
 L-rhamnose recombinant mutant Q156A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 28.3
-
 L-Mannose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 38
-
 L-rhamnose recombinant mutant T191F, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 43
-
 L-Lyxose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 46.3
-
 L-Mannose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 54.8
-
 NADP+ recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 55.5
-
 L-rhamnose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 57
-
 L-rhamnose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 62.2
-
 L-rhamnose recombinant mutant D200H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 62.3
-
 L-Lyxose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 66
-
 NAD+ recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 72.2
-
 NAD+ recombinant mutant S37H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 80.3
-
 L-rhamnose recombinant mutant D200A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 83.5
-
 L-rhamnose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 85.3
-
 L-Lyxose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 130.7
-
 NAD+ recombinant mutant R15T, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 137
-
 L-rhamnose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 308.3
-
 NADP+ recombinant mutant S37H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 355
-
 NADP+ recombinant mutant R15T, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 408.3
-
 L-Lyxose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.378 9
-
 oxidation of L-rhamnose Azotobacter vinelandii

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.378 additional information significant interactions with the 2'-phosphate group of NADP+, but not the 2'-hydroxyl group of NAD+, are consistent with a significant preference for NADP+ over NAD+. Structural insights into coenzyme specificity, overview Azotobacter vinelandii
1.1.1.378 NAD+
-
 Azotobacter vinelandii
1.1.1.378 NADH
-
 Azotobacter vinelandii
1.1.1.378 NADP+
-
 Azotobacter vinelandii
1.1.1.378 NADPH
-
 Azotobacter vinelandii

General Information

EC Number General Information Comment Organism
1.1.1.378 evolution the enzyme belongs to the short-chain dehydrogenase/reductase superfamily Azotobacter vinelandii
1.1.1.378 additional information structure-function analysis, overview. The side chains of Ser146 and Tyr159, Ser148 and Gln156, Thr191, and Asn197 and one water molecular (Wat23) form hydrogen bonds with the hydroxyl groups of C1, C2, C3, and C4 of L-rhamnose, respectively. Wat23 also interacts with the side chains of Asp200, and Lys163 formed hydrogen bond network with the main chains of Ala94 and Asn117 via one water molecular (Wat41). Among these residues, Ser146-Tyr159-Lys163, corresponding to a motif of the catalytic triad, and Ala94 and Asn117 are completely conserved in SDR superfamily enzymes. Phe99 appears to be more important for enzyme catalysis than Ile196 Azotobacter vinelandii
1.1.1.378 physiological function several microorganisms can utilize L-rhamnose as a carbon and energy source through the non-phosphorylative metabolic pathway, in which L-rhamnose 1-dehydrogenase (RhaDH) catalyzes the NAD(P)+-dependent oxidization of Lrhamnose to L-rhamnono-1,4-lactone Azotobacter vinelandii

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.378 0.04
-
 L-Mannose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 0.07
-
 L-Mannose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 0.14
-
 L-rhamnose recombinant mutant Q156A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 0.18
-
 L-Lyxose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 0.31
-
 L-Mannose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 0.41
-
 L-Mannose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 0.86
-
 L-rhamnose recombinant mutant F99A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 1.46
-
 L-rhamnose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 1.46
-
 L-rhamnose recombinant mutant T191F, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 2.1
-
 L-Lyxose recombinant mutant F99Y, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 5.86
-
 L-rhamnose recombinant mutant D200A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 8.05
-
 L-rhamnose recombinant mutant D200H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 12.12
-
 L-rhamnose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 15.8
-
 NADP+ recombinant mutant R15T, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 17.7
-
 L-Lyxose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 18.07
-
 L-Lyxose recombinant mutant I196A, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 23.7
-
 NAD+ recombinant mutant R15T, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 23.9
-
 NADP+ recombinant mutant S37H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 36.5
-
 NAD+ recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 37.4
-
 L-rhamnose recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 47.5
-
 NAD+ recombinant mutant S37H, pH 9.0, 30°C Azotobacter vinelandii
1.1.1.378 282.5
-
 NADP+ recombinant wild-type enzyme, pH 9.0, 30°C Azotobacter vinelandii