CRYSTAL-STRUCTURE OF HUMAN RECOMBINANT ORNITHINE AMINOTRANSFERASE

Ornithine aminotransferase (OAT), a pyridoxal-5'-phosphate dependent e nzyme, catalyses the transfer of the delta-amino group of L-ornithine to 2-oxoglutarate, producing L-glutamate-gamma-semialdehyde, which spo ntaneously cyclizes to pyrroline-5-carboxylate, and L-glutamate. The c rystal structure determination of human recombinant OAT is described i n this paper. As a first step, the structure was determined at low res olution (6 Angstrom) by molecular replacement using the refined struct ure of dialkylglycine decarboxylase as a search model. Crystallographi c phases were then refined and extended in a step-wise fashion to 2.5 Angstrom by cyclic averaging of the electron density corresponding to the three monomers within the asymmetric unit. Interpretation of the r esulting map was straightforward and refinement of the model resulted in an R-factor of 17.1% (X-free = 24.3%). The success of the procedure demonstrates the power of real-space molecular averaging even with on ly threefold redundancy. The alpha(6)-hexameric molecule is a trimer o f intimate dimers with a monomer-monomer interface of 5500 Angstrom(2) per subunit. The three dimers are related by an approximate 3-fold sc rew axis with a translational component of 18 Angstrom, The monomer fo ld is that of a typical representative of subgroup 2 aminotransferases and very similar to those described for dialkylglycine decarboxylase from Pseudomonas cepacia and glutamate-1-semialdehyde aminomutase from Synechococcus. It consists of a large domain that contributes most to the subunit interface, a C-terminal small domain most distant to the 2-fold axis and an N-terminal region that contains a helix, a loop and a three stranded beta-meander embracing a protrusion in the large dom ain of the second subunit of the dimer. The large domain contains the characteristic central seven-stranded beta-sheet (agfedbc) covered by eight helices in a typical alpha/beta fold. The cofactor pyridoxal-5'- phosphate is bound through a Schiff base to Lys292, located in the loo p between strands f and g. The C-terminal domain includes a four-stran ded antiparallel beta-sheet in contact with the large domain and three further helices at the far end of the subunit. The active sites of th e dimer lie, about 25 Angstrom apart, at the subunit and domain interf aces. The conical entrances are on opposite sides of the dimer. In the active site, R180, E235 and R413 are probable substrate binding resid ues. Structure-based sequence comparisons with related transaminases i n this work support that view. In patients suffering from gyrate atrop hy, a recessive hereditary genetic disorder that can cause blindness i n humans, ornithine aminotransferase activity is lacking. A large numb er of frameshift and point mutations in the ornithine aminotransferase gene have been identified in such patients. Possible effects of the v arious point mutations on the structural stability or the catalytic co mpetence of the enzyme are discussed in light of the three-dimensional structure. (C) 1998 Academic Press Limited.