Anatomy of a Simple Acyl Intermediate in Enzyme Catalysis: Combined Biophysical and Modeling Studies on Ornithine Acetyl Transferase

Abstract

Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates.

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

“…This shows the highly flexible nature of these C-terminal residues in the absence of substrates or products bound in the active site of this enzyme. It is worth mentioning here that the stabilization of these three C-terminal residues has also been reported in the acyl-enzyme complex of Sclav OAT, 19 but not in native Sclav OAT. 18 …”

“…The exact role of these C-terminal residues in substrate binding was not established from the acyl-enzyme complex of Sclav OAT, although it has been mentioned that these C-terminal residues might play a role in the binding of the substrate. 19 Our crystal structure of Mtb OAT bound to ORN clearly tells us that there is an important role for these C-terminal residues in substrate binding at the active site. This is clearly evident from the interaction between the side-chain atom N ɛ of ORN and the side-chain atom O γ of Ser404 (Fig.…”

“…11,[14][15][16][17] However, the first crystal structure of an OAT was reported from Streptomyces clavuligerus [S. clavuligerus ornithine acetyltransferase (Sclav OAT)], an organism that is involved in the biosynthesis of clavulanic acid, followed by recent work on the structure of the acyl-enzyme intermediate of this enzyme from the same bacterium. 18,19 Also, the coordinates of the structure from Bacillus halodurans [B. halodurans ornithine acetyltransferase (Bhalo OAT)] have been deposited in the Protein Data Bank (PDB) (PDB code 1VRA). However, the details of the structural work on Bhalo OAT have not been reported so far.…”

“…However, the details of the structural work on Bhalo OAT have not been reported so far. Although recent detailed structural studies on the acyl-enzyme complex of Sclav OAT have discussed a possible catalytic mechanism of OAT during the formation of the acyl-enzyme intermediate, 19 the crystal structures of OAT bound to a substrate analogue or an inhibitor have not been determined so far. With this in view, we have carried out the structure determination of Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT) and its complex with ORN in order to understand the details of the catalytic reaction of this enzyme in Mtb.…”

Amino Acid Biosynthesis

“…Other bacteria use a completely different enzyme for the acetylation chemistry: an ornithine acetyltransferase [138]. This enzyme acts via a sequential Bi Bi kinetic mechanism whereby it accepts an acetyl group from ornithine onto an active-site threonine, generating an acetyl-enzyme intermediate, and then, in a second step, transfers it to glutamate [139]. This route has the advantage of adding and removing the acetyl protecting group using a single enzyme and without paying the metabolic price of acetyl-CoA hydrolysis (although some of the ornithine acetyltransferases can be primed for acetyl donation by using acetyl-CoA as an alternative substrate) [140].…”