Quaternary structure of ornithine aminotransferase in solution and preliminary crystallographic data

Abstract

Ornithine aminotransferase was purified from rat liver and crystallized in the presence of ammonium sulphate and poly(ethy1ene glycol) (PEG 4000). The crystallographic threefold symmetry observed for the resulting two crystal forms stimulated a re-examination of the enzyme's quaternary structure in solution by analytical ultracentrifugation and chemical cross-linking. The results indicate that the oligomeric state or ornithine aminotransferase, under conditions similar to those used in crystallization experiments, is a hexamer (Mr = 256000) rather than a tetramer or higher oligomers as reported previously. The subunits of the enzyme are identical ( M , = 45000).Only the hexagonal prismatic crystals obtained with PEG 4000 were suitable for crystallographic studies and diffracted X-rays to a resolution of at least 0.16 nm. However, these crystals contained an unusual element of disorder which was persistent under a variety of conditions and was only noticeably diminished in the presence of the non-ionic detergent octyl P-glucoside. The crystals apparently belong to the trigonal space group P31 12 (or enantiomorph) with axial lengths of a = 19.5 nm, c = 5.9 nm and contain three monomers per asymmetric unit.Pyridoxal-phosphate-dependent enzymes catalyse a variety of different reactions on amino acid substrates. So far, the three-dimensional structure of only one of these enzymes, aspartate aminotransferase, has been solved by X-ray crystallographic analysis, although several very similar structures have been found for this enzyme prepared from different sources [l -31. We are interested in studying the structure-function relationships of another aminotransferase with quite different specificity, namely ornithine: 2-0x0-glutarate aminotransferase from rat liver. This enzyme transaminates the a-amino group of glutamate, but when presented with ornithine, it transaminates the &amino group exclusively despite the presence of the a-amino group which is configurationally identical with that of glutamate [4]. The mechanism that the enzyme employs to achieve this level of subtlety is not known so its elucidation via determination of the three-dimensional structure is worthwhile.Our preliminary crystallographic studies have been hampered by the fact that although crystals grow readily under a variety of conditions, they contain an element of disorder that, if not eliminated, could prevent a solution of the structure. Although it is clear from extensive earlier work that ornithine aminotransferase is an oligomeric protein

Recent Topics in Pyridoxal 5'-Phosphate Enzyme Studies

“…1990 with that of rat ornithine transcarbamoylase (148), while the sequence around Lys292, which binds PLP, closely resembles that in AspAT (148). Whether the catalytically important residues of AspA T are conserved or not is not clear, for the current structural analysis of OrnAT does not provide any information about the protein folding (149). However, several sequences can be compared to those of AspAT that contain the catalytic residues, i.e.…”

Ornithine Aminotransferase, an Important Glutamate-Metabolizing Enzyme at the Crossroads of Multiple Metabolic Pathways

“…The crystal packing creates a hexamer formed by a trimer of dimers related by an approximately 3-fold screw axis. Although it is not possible to tell from the crystal structure whether the hexamer is of biological relevance or merely an effect of crystal packing, there is experimental evidence for the existence of hexamers of rat liver OAT in solution approaching crystallization conditions [61]. Assemblies of dimers or tetramers have been observed in electron microscopic studies on pig kidney OAT [62], and oligomers are detected in high concentrations of mouse liver OAT [63].…”

Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase

“…Nevertheless, analytical ultracentrifugation studies on crude extract of rat liver have suggested that OAT presents a molecular weight dependent on the enzyme concentration assuming a two-step aggregation of the monomer forming at first trimers (130–140 kDa) and then hexamers (280 kDa) [17]. Crystallographic studies on purified rat liver OAT [18] and, more recently, on human recombinant OAT (hOAT) both ligand-free [19] and in complex with different substrate analogues [20, 21], indicated that the human enzyme could also assume an hexameric assembly composed by three homodimers hold together mainly by electrostatic interactions. Moreover, on the basis of the hOAT crystal structure, the dimeric unit of the enzyme belongs to the fold type I class of PLP-enzymes and it was proposed that the dimer could represent the functional unit of the enzyme [19].…”