3-DIMENSIONAL STRUCTURE OF MESO-DIAMINOPIMELIC ACID DEHYDROGENASE FROM CORYNEBACTERIUM-GLUTAMICUM

Diaminopimelate dehydrogenase catalyzes the NADPH-dependent reduction of ammonia and L-2-amino-6-ketopimelate to form meso-diaminopimelate, the direct precursor of L-lysine in the bacterial lysine biosynthetic pathway. Since mammals lack this metabolic pathway, inhibitors of enzy mes in this pathway may be useful as antibiotics or herbicides. Diamin opimelate dehydrogenase catalyzes the only oxidative deamination of an amino acid of D configuration and must additionally distinguish betwe en two chiral amino acid centers on the same symmetric substrate. The Corynebacterium glutamicum enzyme has been cloned, expressed in Escher ichia coli, and purified to homogeneity using standard biochemical pro cedures [Reddy, S. G., Scapin, G., & Blanchard, J. S. (1996) Proteins: Structure, Funct. Genet. 25, 514-516], The three-dimensional structur e of the binary complex of diaminopimelate dehydrogenase with NADP(+) has been solved using multiple isomorphous replacement procedures and noncrystallographic symmetry averaging. The resulting model has been r efined against 2.2 A diffraction data to a conventional crystallograph ic R-factor of 17.0%. Diaminopimelate dehydrogenase is a hamodimer of structurally not identical subunits. Each subunit is composed of three domains. The N-terminal domain contains a modified dinucleotide bindi ng domain, or Rossman fold (six central beta-strands in a 213456 topol ogy surrounded by five alpha-helices). The second domain contains two alpha-helices and three beta-strands. This domain is referred to as th e dimerization domain, since it is involved in forming the monomer-mon omer interface of the dimer. The third or C-terminal domain is compose d of six beta-strands and five alpha-helices. The relative position of the N- and C-terminal domain in the two monomers is different, defini ng an open and a closed conformation that may represent the enzyme's b inding and active state, respectively. In both monomers the nucleotide is bound in an extended conformation across the C-terminal portion of the beta-sheet of the Rossman fold, with its C4 facing the C-terminal domain. In the closed conformer two molecules of acetate have been re fined in this region, and we postulate that they define the DAP bindin g site. The structure of diaminopimelate dehydrogenase shows interesti ng similarities to the structure of glutamate dehydrogenase [Baker, P. J., Britton, K. L., Rice, D. W., Rob, A,, & Stillmann, T. J. (1992a) J. Mel. Biol. 228, 662-671] and leucine dehydrogenase [Baker, P. J., T urnbull, A. P., Sedelnikova, S. E., Stillman, T. J., & Rice, D. W. (19 95) Structure 3, 693-705] and also resembles the structure of dihydrod ipicolinate reductase [Scapin, G., Blanchard, J. S., & Sacchettini, J. C. (1995) Biochemistry 34, 3502-3512], the enzyme immediately precedi ng it in the diaminopimelic acid/lysine biosynthetic pathway.