High‐resolution Electron Microscopic Studies on the Quaternary Structure of Ornithine Aminotransferase from Pig Kidney

Abstract

The ornithine aminotransferase (OAT) from pig kidney has been studied on the basis of highresolution electron microscopy and the morphological appearance of the apoenzyme and holoenzyme have been examined. The quaternary structure of the OAT molecules in the presence of 5 mM pyridoxal 5'-phosphate could be established. The enzyme molecule appears to be built up of two morphological units, called M1. The native holoenzyme, termed morphological unit M2, measures . 10.9 nm in length and 5.8 nm in width and its molecular mass is approximately 168 kDa, based on electron microscopical calculations. Since the enzyme is composed of only one type of 45-kDa subunit, the holoenzyme is a homotetramer. Each M1 is composed of two subunits and, as seen in top-view projection, has an oval to triangular shape. Upon tilting to 40" the triangular shape changes into three distinct centers of mass. This morphological differentiation reflects the inner organization of M1, i.e. the shape of the individual subunit deviates from strictly globular proteins. This observation is compatible with the notion that the 45-kDa subunit consists of one large and one small domain. By tilting to 40", both large domains in M1 represent two of the three centers of mass, while the third center of mass is attributed to the superposition of both small domains. Thus, the four domains of both subunits in M1, in accordance with the triangular top-view projection, are quasi-tetrahedrally arranged. Since the change in shape of M1 upon tilting is only obvious in one of the two halves of the native OAT, it suggests that both morphological units of M2 are oriented asymmetrically relative to one another.

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

“…Moreover, we demonstrated that both holo and apo exist in solution as a tetramer. Although these data confirm previous analyses on mammalian OATs purified from different sources [13–16], they are in contrast with crystallographic analyses indicating the hexameric assembly of the enzyme [18–21]. It is possible that the crystallization conditions, known to increase the solute concentration over that expected for an ideal solution, could promote the hexamerization.…”

“…In fact ultracentrifugation and SEC studies on the OAT purified from rat and human liver [13–15] indicated that the enzyme has a tetrameric assembly. Accordingly, high resolution electron microscopy studies on OAT purified from pig kidney indicated that the holoenzyme is a homotetramer [16]. Moreover, Sanada et al have reported that the apo-OAT exists in dimeric form [14] raising a question about the possible role of the PLP in the OAT oligomerization.…”

Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA

“…Testing the consistency between the proteome profiles of both PRLTS3 patient fibroblast cultures (in triplicates) versus ClpP-null MEFs, we also noted significant accumulations of eleven mitochondrial matrix proteins ( Figure 4 C), which are not known as interactors of ClpX or ClpB: OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, see Table 2 . Importantly, all of them are known for their assembly into homo-multimers or heterodimers [ 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. OAT, PC, ALDH2, PMPCB, and ACADSB also showed significant accumulation in ClpP-null brains ( Table S3 ).…”

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

“…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]. The oligomerization state seems to change the kinetic properties of the enzyme [30], and may partly explain the differences observed between tissues, although the kinetic studies performed up till now have never shown any sign of cooperation between the various subunits (the kinetics seems to be of the Michaelis Menton type, see above).…”