Literature summary for 1.1.1.378 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 .

Search for more literature for 1.1.1.378

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
D200A	site-directed mutagenesis, the mutant shows 16% activity with L-rhamnose compared to wild-type	Azotobacter vinelandii
D200H	site-directed mutagenesis, the mutant shows 22% activity with L-rhamnose compared to wild-type	Azotobacter vinelandii
F99A	site-directed mutagenesis, the mutant is inactive	Azotobacter vinelandii
F99Y	site-directed mutagenesis, the mutant is inactive	Azotobacter vinelandii
I196A	site-directed mutagenesis, the mutant is inactive	Azotobacter vinelandii
Q156A	site-directed mutagenesis, the mutant is almost inactive	Azotobacter vinelandii
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
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
T191F	site-directed mutagenesis, the mutant shows 3.9% activity with L-rhamnose compared to wild-type	Azotobacter vinelandii

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Azotobacter vinelandii	-	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
L-lyxose + NAD(P)+	-	Azotobacter vinelandii	? + NAD(P)H + H+	-	?
L-mannose + NAD(P)+	-	Azotobacter vinelandii	? + NAD(P)H + H+	-	?
L-rhamnose + NAD+	L-rhamnose is the best substrate, preference for NADP+ over NAD+	Azotobacter vinelandii	L-rhamnono-1,4-lactone + NADH + H+	-	?
L-rhamnose + NADP+	-	Azotobacter vinelandii	L-rhamnono-1,4-lactone + NADPH + H+	-	?
L-rhamnose + NADP+	L-rhamnose is the best substrate, preference for NADP+ over NAD+	Azotobacter vinelandii	L-rhamnono-1,4-lactone + NADPH + H+	-	?
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

Subunits	Comment	Organism
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

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism
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
NAD+	-	Azotobacter vinelandii
NADH	-	Azotobacter vinelandii
NADP+	-	Azotobacter vinelandii
NADPH	-	Azotobacter vinelandii

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the short-chain dehydrogenase/reductase superfamily	Azotobacter vinelandii
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
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]

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