Structures of N‐acetylornithine transcarbamoylase from Xanthomonas campestris complexed with substrates and substrate analogs imply mechanisms for substrate binding and catalysis

Abstract

N-acetyl-L-ornithine transcarbamoylase (AOTCase) is a new member of the transcarbamoylase superfamily that is essential for arginine biosynthesis in several eubacteria. We report here crystal structures of the binary complexes of AOTCase with its substrates, carbamoyl phosphate (CP) or N-acetyl-L-ornithine (AORN), and the ternary complex with CP and N-acetyl-L-norvaline. Comparison of these structures demonstrates that the substrate-binding mechanism of this novel transcarbamoylase is different from those of aspartate and ornithine transcarbamoylases, both of which show ordered substrate binding with large domain movements. CP and AORN bind to AOTCase independently, and the main conformational change upon substrate binding is ordering of the 80's loop, with a small domain closure around the active site and little movement of the 240's loop. The structures of the complexes provide insight into the mode of substrate binding and the mechanism of the transcarbamoylation reaction.

Stabilizing effect of knots on proteins

“…On the other hand in the AOTCase pathway, the 2 substrates are bound independently. This process involves small reordering of the 80's loop, small-domain closure around the active site, and a small translocation of the 240's loop (17).…”

“…OTCase and AOTCase, participate in the arginine biosynthetic pathway, but the presence of the knot in AOTCase makes the corresponding pathway distinct (17). Both proteins contain two active sites-the first binds carbonyl phosphatase (CP), whereas the second site (which is modified by the knot structure) binds either N-acetylornithine or L-ornithine, in the case of 1yh1 and 1c9y, respectively.…”

“…The OTCase pathway shows ordered two-substrate binding with large domain movements, whereas in the AOTCase pathway the two substrates are bound independently with small reordering of the 80's loop, small domain closure around the active site, and a small translocation of the 240's loop (17). Thus, it seems that the knot plays two roles here: it changes the environment for the second substrate N-acetylcitrulline binding, and-as shown in this articlemakes the structure more stable.…”

Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

“…1, Box 1). The enzyme ArgFЈ (EC 2.1.3.9) is a distant homologue of the classical OTC (EC 2.1.3.3) but lacks an OTC-specific motif, the SMG motif, and the substrate-binding mechanism of this enzyme appears to be different from that of both ornithine and aspartate carbamoyltransferases (68). X. campestris AOTC is presently the only carbamoyltransferase known to catalyze this reaction in the pure form, but genes similar to argFЈ, also lacking the SMG motif, were identified in other species: X. axonopodis, Xylella fastidiosa, Bacteroides thetaiotaomicron, Cytophaga hutchinsonii, Tannerella forsythensis, and Prevotella ruminicola (50).…”

Knotoids, Braidoids and Applications

“…The graphics are generated using VMD [25]. The analyzed protein is the N-acetylornithine protein (PDB entry: 3KZN) [26]. The structure was downloaded from the Protein Database (PDB) [27].…”