Literature summary for 2.6.1.30 extracted from

Yoshikane, Y.; Yokochi, N.; Yamasaki, M.; Mizutani, K.; Ohnishi, K.; Mikami, B.; Hayashi, H.; Yagi, T.
Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099 (2008), J. Biol. Chem., 283, 1120-1127.

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Mesorhizobium loti

Crystallization (Commentary)

Crystallization (Comment)	Organism
native enzyme, space group P43212, diffraction to 2.0 A resolution. Complexes with pyridoxamine, pyridoxal, and pyridoxyl-L-alanine at 1.7 A, 1.7 A, and 2.0 A resolution, respectively. Enzyme is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys197 in the active site. The carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg336 and Arg345	Mesorhizobium loti

Crystallization (Comment)

Organism

native enzyme, space group P43212, diffraction to 2.0 A resolution. Complexes with pyridoxamine, pyridoxal, and pyridoxyl-L-alanine at 1.7 A, 1.7 A, and 2.0 A resolution, respectively. Enzyme is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven beta-sheets and eight alpha-helices, and a smaller C-terminal domain, consisting of three beta-sheets and four alpha-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys197 in the active site. The carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg336 and Arg345

Mesorhizobium loti

Protein Variants

Protein Variants	Comment	Organism
E68A	low pyridoxamine 5'-phosphate:pyruvate aminotransferase activity, decrease in activity towards pyridoxamine	Mesorhizobium loti
E68G	low pyridoxamine 5'-phosphate:pyruvate aminotransferase activity, decrease in activity towards pyridoxamine	Mesorhizobium loti
R336A	significant decrease in affinity for pyruvate	Mesorhizobium loti

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.044	-	pyridoxamine	wild-type, pH 9.0, 30°C	Mesorhizobium loti
0.1	-	pyridoxamine	mutant R336A, pH 9.0, 30°C	Mesorhizobium loti
0.34	-	pyruvate	wild-type, pH 9.0, 30°C	Mesorhizobium loti
1.3	-	pyridoxamine	mutant E68A, pH 9.0, 30°C	Mesorhizobium loti
2.5	-	pyridoxamine	mutant E68G, pH 9.0, 30°C	Mesorhizobium loti
3.4	-	pyridoxamine 5'-phosphate	mutant E68A, pH 9.0, 30°C	Mesorhizobium loti
6.9	-	pyruvate	mutant R336A, pH 9.0, 30°C	Mesorhizobium loti

Organism

Organism	UniProt	Comment	Textmining
Mesorhizobium loti	Q988B8	-	-
Mesorhizobium loti MAFF303099	Q988B8	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
pyridoxamine + pyruvate = pyridoxal + L-alanine	enzyme first binds pyridoxamine and then forms a Michaelis complex with the incoming pyruvate. The ketimine is formed through nucleophilic attack of the N-4' atom of pyridoxamine on the alpha-carbon atom of pyruvate, which is followed by the release of a water molecule. The stereospecific 1,3-prototropic shift between the ketimine and external aldimine via the quinonoid intermediate is accomplished through general base catalysis by Lys197. Pyridoxal and L-alanine are formed from the external aldimine and released from enzyme	Mesorhizobium loti	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
pyridoxamine + pyruvate	-	Mesorhizobium loti	pyridoxal + L-alanine	-	?
pyridoxamine + pyruvate	-	Mesorhizobium loti MAFF303099	pyridoxal + L-alanine	-	?
pyridoxamine 5'-phosphate + pyruvate	-	Mesorhizobium loti	pyridoxal 5'-phosphate + L-alanine	-	?
pyridoxamine 5'-phosphate + pyruvate	-	Mesorhizobium loti MAFF303099	pyridoxal 5'-phosphate + L-alanine	-	?

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.062	-	pyridoxamine 5'-phosphate	mutant E68G, pH 9.0, 30°C	Mesorhizobium loti
0.22	-	pyridoxamine 5'-phosphate	mutant E68A, pH 9.0, 30°C	Mesorhizobium loti
0.77	-	pyridoxamine	mutant E68G, pH 9.0, 30°C	Mesorhizobium loti
0.78	-	pyridoxamine	mutant E68A, pH 9.0, 30°C	Mesorhizobium loti
2	8	pyridoxamine	mutant R336A, pH 9.0, 30°C	Mesorhizobium loti
2	8	pyridoxamine	wild-type, pH 9.0, 30°C	Mesorhizobium loti

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	the bulky side chain of Glu68 interferes with the binding of the phosphate moiety of pyridoxal 5'-phosphate and makes the enzyme specific to pyridoxal	Mesorhizobium loti