A Conserved Lysine in β‐Lactam Synthetase Assists Ring Cyclization: Implications for Clavam and Carbapenem Biosynthesis

Abstract

β-Lactam synthetase (β-LS) is the paradigm of a growing class of enzymes that form the critical β-lactam ring in the clavam and carbapenem antibiotics. β-LS catalyzes a two stage reaction in which N2-(2-carboxyethyl)-l-arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to β-lactam formation is high, and that the overall rate of reaction is partially limited to a protein conformational change rather than to the chemical step alone of closing the strained ring. β-Lactam formation was evaluated on the basis of X-ray crystal structures, site-specific mutation, and kinetic and computational studies. The combined evidence clearly points to a reaction coordinate involving the formation of a tetrahedral transition state/intermediate stabilized by a conserved Lys. The combination of substrate pre-organization, a well-stabilized transition state and an excellent leaving group facilitates this acyl substitution to account for the strong forward commitment to catalysis and to lower the barrier of four-membered ring formation to the magnitude of a protein conformational change.

Enantiomerically Pure trans-β-Lactams from α-Amino Acids via Compact Fluorescent Light (CFL) Continuous-Flow Photolysis

“…The results are consistent with a mechanism involving a stereospecific intramolecular Wolff rearrangement with full retention of absolute configuration . Intramolecular attack by the trityl-protected amine on the intermediate ketene A , formally a disfavored 4-exo-dig process under Baldwin’s rules, − affords the product β-lactam. Other key observations from this work were (1) the stability and crystallinity of imidazolides 1 , which translate into handling ease in the sequence; (2) the observation of 3 as the major isolable product from rhodium(II) catalysis, silver(I) catalysis, and light (somewhat rare, fully expected, and best yields, respectively); and (3) the stereospecificity of the Wolff rearrangement, as gauged by the clean formation of ent - 3 when starting with d -serine-derived ent - 1 .…”

Stereoselective Production of Dimethyl-Substituted Carbapenams via Engineered Carbapenem Biosynthesis Enzymes

“…The model is built on the basis of the acyl-adenylate N 2 -(2-carboxymethyl)­arginine-AMP species bound in a crystal structure of β-LS (PDB 1MBZ). The Lys443 residue is proposed to assist in ring cyclization via stabilization of the proposed “tetrahedral” intermediate (resulting upon nucleophile attack by the secondary amine of t -CMP). The shown Tyr345-Glu380 dyad is proposed to deprotonate the amine involved in the intramolecular β-lactam formation …”

Origins of the β-lactam rings in natural products

“…The Bls reaction begins with activation of the substrate, carboxyethylarginine, by aminoadenylation using ATP, followed by closure of the b-lactam ring to give the monocyclic intermediate, deoxyguanidinoproclavaminic acid, with concomitant release of pyrophosphate (PPi) and AMP. [52][53][54] Therefore, the closure of the clavam b-lactam ring occurs by amide bond formation and is essentially a reversal of the b-lactam cleavage reaction caused by b-lactamases. 51 This reaction is very different from b-lactam ring formation by IPNS, where the b-lactam amide linkage is preexisting in the ACV substrate.…”