Literature summary for 1.1.1.B66 extracted from

Guo, J.; Higgins, M.A.; Daniel-Ivad, P.; Ryan, K.S.
An asymmetric reductase that intercepts acyclic imino acids produced in situ by a partner oxidase (2019), J. Am. Chem. Soc., 141, 12258-12267 .

Search for more literature for 1.1.1.B66

Cloned(Commentary)

Cloned (Comment)	Organism
gene bsp5, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant overexpression of His6-tagged wild-type and mutant enzymes from pUC57-Bsp5 vector in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha	Bacillus sp. 5mfcol3.1

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme Bsp5 in complex with D-arginine and coenzyme NADPH, hanging drop vapour diffusion, mixing of 800 nl of 10 mg/m protein in 20 mM HEPES, 50 mM NaCl, pH 7.5, 5 mM D-Arg, and 5 mM NADPH, with 800 nl of reservoir solution containing 20% w/v PEG 3350 and 0.2 M sodium tartrate dibasic, equilibration against 0.05 ml of reservoir solution, room temperature, 10 days, method optimization, X-ray diffraction structure determination and analysis at 1.6 A resolution, molecular replacement, modeling	Bacillus sp. 5mfcol3.1

Crystallization (Comment)

Organism

purified enzyme Bsp5 in complex with D-arginine and coenzyme NADPH, hanging drop vapour diffusion, mixing of 800 nl of 10 mg/m protein in 20 mM HEPES, 50 mM NaCl, pH 7.5, 5 mM D-Arg, and 5 mM NADPH, with 800 nl of reservoir solution containing 20% w/v PEG 3350 and 0.2 M sodium tartrate dibasic, equilibration against 0.05 ml of reservoir solution, room temperature, 10 days, method optimization, X-ray diffraction structure determination and analysis at 1.6 A resolution, molecular replacement, modeling

Bacillus sp. 5mfcol3.1

Protein Variants

Protein Variants	Comment	Organism
H130A	site-directed mutagenesis, the mutant shows highly reduced activity with substrate (4E)-5-carbamimidamido-2-iminopent-4-enoic acid, but unaltered activity with 5-carbamimidamido-2-iminopentanoic acid compared to wild-type	Bacillus sp. 5mfcol3.1
H130A/Y132F	site-directed mutagenesis, the mutant shows highly reduced activity with substrate (4E)-5-carbamimidamido-2-iminopent-4-enoic acid, but increased activity with 5-carbamimidamido-2-iminopentanoic acid compared to wild-type	Bacillus sp. 5mfcol3.1
additional information	the mutations do not impact dimerization	Bacillus sp. 5mfcol3.1
R229A	site-directed mutagenesis, the mutant shows reduced activity with substrates (4E)-5-carbamimidamido-2-iminopent-4-enoic acid and 5-carbamimidamido-2-iminopentanoic acid compared to wild-type	Bacillus sp. 5mfcol3.1
Y132F	site-directed mutagenesis, the mutant shows highly reduced activity with substrate (4E)-5-carbamimidamido-2-iminopent-4-enoic acid, but unaltered activity with 5-carbamimidamido-2-iminopentanoic acid compared to wild-type	Bacillus sp. 5mfcol3.1
Y97F	site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme	Bacillus sp. 5mfcol3.1

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
70000	-	about, recombinant His-tagged enzyme, gel filtration	Bacillus sp. 5mfcol3.1

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Bacillus sp. 5mfcol3.1	enzyme Bsp5 reduces acyclic imino acids produced in situ by a partner oxidase	?	-	-

Organism

Organism	UniProt	Comment	Textmining
Bacillus sp. 5mfcol3.1	A0A1I4FUG4	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, gel filtration, and ultrafiltration	Bacillus sp. 5mfcol3.1

Reaction

Reaction	Comment	Organism	Reaction ID
an acyclic iminoacid + NADPH + H+ = an amino acid + NADP+	no imine reducing activity on cyclic compounds	Bacillus sp. 5mfcol3.1	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(4E)-5-carbamimidamido-2-iminopent-4-enoic acid + NADPH + H+	-	Bacillus sp. 5mfcol3.1	D-2-dehydroarginine + NADP+	-	?
5-carbamimidamido-2-iminopentanoic acid + NADPH + H+	Bsp5 shows poor activity in the reverse reaction direction	Bacillus sp. 5mfcol3.1	D-arginine + NADP+	-	?
additional information	enzyme Bsp5 reduces acyclic imino acids produced in situ by a partner oxidase	Bacillus sp. 5mfcol3.1	?	-	-
additional information	the asymmetric reductase intercepts acyclic imino acids produced in situ by a partner oxidase (Ind4 from Paenibacillus sp.) in a coupled assay, overview. Nonenzymatic hydrolysis of acyclic imino acids gives compounds (4E)-5-carbamimidamido-2-oxopent-4-enoic acid and 5-carbamimidamido-2-oxopentanoic acid. Bsp5 fails to reduce 5-carbamimidamido-2-oxopentanoic acid, and incubation of Bsp5 with 2-methylpyrroline also results in no reaction. Bsp5 lacks imine reducing activity on cyclic substrates. LC-MS analysis of the molecules	Bacillus sp. 5mfcol3.1	?	-	-

Subunits

Subunits	Comment	Organism
homodimer	2 * 35939, recombinant wild-type enzyme, sequence calculation and mass spectrometry	Bacillus sp. 5mfcol3.1
More	dimer three-dimensional structure modeling with dimeric interface, overview	Bacillus sp. 5mfcol3.1

Synonyms

Synonyms	Comment	Organism
asymmetric reductase	-	Bacillus sp. 5mfcol3.1
BSp5	-	Bacillus sp. 5mfcol3.1
imino acid reductase	-	Bacillus sp. 5mfcol3.1

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Bacillus sp. 5mfcol3.1

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Bacillus sp. 5mfcol3.1

Cofactor

Cofactor	Comment	Organism	Structure
NADP+	-	Bacillus sp. 5mfcol3.1
NADPH	-	Bacillus sp. 5mfcol3.1

General Information

General Information	Comment	Organism
evolution	enzyme Bsp5 belongs to the D-2-hydroxyacid dehydrogenase family	Bacillus sp. 5mfcol3.1
additional information	structure-based mechanism of Bsp5's imine reductase activity	Bacillus sp. 5mfcol3.1
physiological function	Bsp5 is an imino acid reductase from the D-2-hydroxyacid dehydrogenase family that reduces acyclic imino acids produced in situ by a partner oxidase	Bacillus sp. 5mfcol3.1