Crystallographic Study of Steps along the Reaction Pathway of d-Amino Acid Aminotransferase^,

Abstract

The three-dimensional structures of two forms of the D-amino acid aminotransferase (D-aAT) from Bacillus sp. YM-1 have been determined crystallographically: the pyridoxal phosphate (PLP) form and a complex with the reduced analogue of the external aldimine, N-(5'-phosphopyridoxyl)-d-alanine (PPDA). Together with the previously reported pyridoxamine phosphate form of the enzyme [Sugio et al. (1995) Biochemistry 34, 9661], these structures allow us to describe the pathway of the enzymatic reaction in structural terms. A major determinant of the enzyme's stereospecificity for D-amino acids is a group of three residues (Tyr30, Arg98, and His100, with the latter two contributed by the neighboring subunit) forming four hydrogen bonds to the substrate alpha-carboxyl group. The replacement by hydrophobic groups of the homologous residues of the branched chain L-amino acid aminotransferase (which has a similar fold) could explain its opposite stereospecificity. As in L-aspartate aminotransferase (L-AspAT), the cofactor in D-aAT tilts (around its phosphate group and N1 as pivots) away from the catalytic lysine 145 and the protein face in the course of the reaction. Unlike L-AspAT, D-aAT shows no other significant conformational changes during the reaction.

Pre-steady-state Reaction of 5-Aminolevulinate Synthase

“…Closure of the enzyme around the substrate appears to function primarily to increase substrate specificity. Interestingly, D-amino acid aminotransferase, a PLP-dependent enzyme that does not undergo significant conformational changes during reaction, has a broad substrate specificity (29,30). ALAS, however, shows strict substrate specificity for glycine; no other naturally occurring amino acid has been found to act as a substrate (31).…”

Structural and Functional Differences in Two Cyclic Bacteriocins with the Same Sequences Produced by Lactobacilli

“…Many peptides in peptidoglycan of the bacterial cell wall contain D-Ala and D-Glu residues, which are constructed by D-amino acid transaminase (27,30). Bacterial racemases for Ala, Arg, Glu, Phe, and Pro have been reported (1,3).…”

Structural Basis for D-Amino Acid Transamination by the Pyridoxal 5′-Phosphate-dependent Catalytic Antibody 15A9

“…3B) and D-amino acid aminotransferase from Bacillus species YM-1 (DaAT) in complex with PPL-D-alanine (26) (Fig. 3C).…”

“…The ⑀-amino group of the catalytic base Lys-258 is equidistant from atoms C4Ј and C␣ (3.4 and 3.6 Å, respectively) and is located on the si face of the coenzyme adduct. C, DaAT in complex with PPL-D-alanine (Protein Data Bank code 3DAA) (26). The pyridine ring of the cofactor is engaged in stacking interactions with Leu-201 and the backbone of Ser-180.…”