X-RAY STUDIES ON THE STRUCTURE, SPECIFICITY AND MECHANISM OF THE OPINE DEHYDROGENASE ENZYME SUPERFAMILY. ¹S.J. Langridge, ¹K.L. Britton, ²Y. Asano and ¹D.W. Rice, ¹Krebs Institute, University of Sheffield, Sheffield, S10 2TN, UK and ²Biotechnology Research Center, Toyama Prefectual University, Toyama 939-03, Japan.

The structure of a novel opine dehydrogenase from Arthrobacter has been solved by isomorphous replacement and provides insights into the mechanism and substrate specificity of the enzyme superfamily to which it belongs. Crown gall opines are the products of the NAD(P)H-dependent reductive condensation between an (-keto acid and the (- or (-NH₂ group of an amino acid in a reaction catalysed by a family of enzymes, generically referred to as the opine dehydrogenases (reviewed in Thompson, J. and Donkersloot, J.A. Annu. Rev. Biochem. 1992 61 517-557). The enzymes catalysing this chemistry are encoded on large plasmids resident in virulent strains of Agrobacterium. These tumour inducing plasmids are required for crown gall induction and tumourogenesis involves the excision of a segment of the plasmid DNA on which the opine dehydrogenase gene is located. Following integration of the DNA into the plant genome, the plant cell machinery is hijacked to divert resources to the synthesis of opines which permit growth of the tumour. Sequence studies have established that opine dehydrogenases belong to an enzyme superfamily with differential specificity for the keto acid and amino acid partners. Recently, the gene for a novel opine dehydrogenase from Arthrobacter has been sequenced and shown to have 30% sequence identity with the octapine dehydrogenase from Agrobacterium tumefaciens. This enzyme is a homodimer of subunit Mr 70,000 and has been overexpressed in E. coli and crystallised. X-ray analysis shows that the crystals which diffract to beyond 1.8 belong to the orthorhombic space group P2₁2₁2 with a = 104, b = 79 and c = 45 with a single monomer in the asymmetric unit. The structure will be described and insights into the catalytic mechanism and the differential substrate specificity will be discussed.