The structures of the C146A variant of the amidase from Pyrococcus horikoshii bound to glutaramide and acetamide suggest the basis of amide recognition.

The nitrilase superfamily enzymes from Pyrococcus abyssi and Pyrococcus horikoshii hydrolyze several different amides. No nitriles that we tested were hydrolyzed by either enzyme. Propionamide and acetamide were the most rapidly hydrolyzed of all the substrates tested. Amide substrate docking studies on the wild-type and C146A variant P. horikoshii enzymes suggest a sequence in which the incoming amide substrate initially hydrogen bonds to the amino group of Lys-113 and the backbone carbonyl of Asn-171. When steric hindrance is relieved by replacing the cysteine with alanine, the amide then docks such that the amino group of Lys-113 and the backbone amide of Phe-147 are hydrogen-bonded to the substrate carbonyl oxygen, while the backbone carbonyl oxygen of Asn-171 and the carboxyl oxygen of Glu-42 are hydrogen-bonded to the amino group of the substrate. Here, we confirm the location of the acetamide and glutaramide ligands experimentally in well-resolved crystal structures of the C146A mutant of the enzyme from P. horikoshii. This ligand location suggests that there is no direct interaction between the substrate amide and the other active site glutamate, Glu-120, and supports an active-site geometry leading to the formation of the thioester intermediate via an attack on the si-face of the amide by the sulfhydryl of the active site cysteine.

A new crystal form of MshB from Mycobacterium tuberculosis with glycerol and acetate in the active site suggests the catalytic mechanism.

MshB, a zinc-based deacetylase, catalyses a step in the mycothiol biosynthetic pathway that involves the deacetylation of 1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcNAc-Ins), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcN-Ins) and acetate. In this study, MshB was expressed, purified and crystallized. A new crystal form was encountered in 0.1 M sodium acetate, 0.2 M ammonium sulfate, 25% PEG 4000 pH 4.6. The crystals diffracted to 1.95 Å resolution and the resulting electron-density map revealed glycerol and the reaction product, acetate, in the active site. These ligands enabled the natural substrate GlcNAc-Ins to be modelled in the active site with some certainty. One acetate O atom is hydrogen bonded to Tyr142 and is located 2.5 Å from the catalytic zinc. The other acetate O atom is located 2.7 Å from a carboxylate O atom of Asp15. This configuration strongly suggests that Asp15 acts both as a general base catalyst in the nucleophilic attack of water on the amide carbonyl C atom and in its protonated form acts as a general acid to protonate the amide N atom. The configuration of Tyr142 differs from that observed previously in crystal structures of MshB (PDB entries 1q74 and 1q7t) and its location provides direct structural support for recently published biochemical and mutational studies suggesting that this residue is involved in a conformational change on substrate binding and contributes to the oxyanion hole that stabilizes the tetrahedral intermediate.