Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | 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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Mesorhizobium loti | Q988B8 | - |
- |
Mesorhizobium loti MAFF303099 | Q988B8 | - |
- |
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 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | 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 |