DETERMINATION OF THE STRUCTURE OF ALANINE RACEMASE FROM BACILLUS-STEAROTHERMOPHILUS AT 1.9-ANGSTROM RESOLUTION

The molecular structure of alanine racemase from Bacillus stearothermo philus was determined by X-ray crystallography to a resolution of 1.9 Angstrom. The alanine racemase monomer is composed of two domains, an eight-stranded alpha/beta barrel at the N-terminus, which includes res idues 1-240, and a C-terminal domain essentially composed of beta-stra nd (residues 241-388). In the structure of the dimer the mouth of the alpha/beta barrel of one monomer faces the second domain of the other monomer. The pyridoxal 5'-phosphate (PLP) cofactor lies in and above t he mouth of the alpha/beta barrel and is covalently linked via an aldi mine linkage to Lys39, which is at the C-terminus of the first beta-st rand of the alpha/beta barrel. This is the first example of a PLP cofa ctor binding in the active site of a alpha/beta barrel. A number of ot her residues are involved in maintaining the position of the PLP in th e protein. Of these, Arg219 is the most interesting, as it forms a hyd rogen bond with the pyridine nitrogen of the cofactor. This is the fir st known occurrence of such an interaction with PLP and is expected to influence the electron delocalization in the PLP-alanine intermediate s. A second arginine residue, Arg136, donates a hydrogen bond to the p henolic oxygen of PLP and may be involved in the binding of substrate as well as stabilization of intermediates. Finally, Tyr265', from the second monomer, is postulated to be 2 proton donor to the carbanion in termediate.