Stereospecificity of Thermostable Ornithine 5-Aminotransferase for the Hydrogen Transfer in the L- and D-Ornithine Transamination (MOLECULAR BIOFUNCTION-Molecular Microbial Science)

Abstract

The thermostable ornithine 5-aminotransferase of a thermophile, Bacillus sp. YM-2 is unique in acting on both enantiomers of ornithine, although less effectively on the D-enantiomer. We studied the stereospecificity of the enzyme for the hydrogen abstraction from C-5 of the substrate moiety and the addition and removal of the hydrogen at C-4' of the cofactor (pyridoxal phosphate and pyridoxamine phosphate) moiety of the external Schiff base intermediate in the transamination of L- and D-ornithine. [5- 3H]L- and D-ornithines were prepared by incubation of L- and D-ornithines with the B. sp. YM-2 ornithine 5-aminotransferase in 3H2O, respectively. The C-5 of the tritiated L-and D-ornithine was proved to have the S-configuration with L-ornithine 5-aminotransferase of a mesophile, Bacillus sphaericus, catalyzing the stereospecific abstraction of pro-S hydrogen from C-5 of L-ornithine and amino acid racemase with lowsubstrate specificity of Pseudomonas putida . When apo-form of the enzyme was incubated with pyridoxamine 5'-phosphates that was stereospecifically tritiated at C-4' and 2-oxoglutarate in the presence of L-ornithine or D-ornithine, tritium was released exclusively from (4'S)-[4'-3H] pyridoxamine. These results suggest that the B. sp. YM-2 ornithine 5-aminotransferase stereospecifically abstracts the pro-S hydrogen from C-5 of L- and D-ornithine. The hydrogen abstracted is then transferred to C-4' of the cofactor moiety stereospecifically on the si face of the external Schiff base intermediate irrespective of the C-2 configuration of amino donor.