Mechanistic Insights from the Crystal Structure of Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase Complexed with the Adenylate Intermediate

Abstract

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase, or MenE, catalyzes an essential step in vitamin K biosynthesis and is a valuable drug target. Like many other adenylating enzymes, it changes its structure to accommodate substrate binding, catalysis, and product release along the path of a domain alternation catalytic mechanism. We have determined the crystal structure of its complex with the adenylation product, o-succinylbenzoyl-adenosine monophosphate (OSB-AMP), and captured a new postadenylation state. This structure presents unique features such as a strained conformation for the bound adenylate intermediate to indicate that it represents the enzyme state after completion of the adenylation reaction but before release of the C domain in its transition to the thioesterification conformation. By comparison to the ATP-bound preadenylation conformation, structural changes are identified in both the reactants and the active site to allow inference about how these changes accommodate and facilitate the adenylation reaction and to directly support an in-line backside attack nucleophilic substitution mechanism for the first half-reaction. Mutational analysis suggests that the conserved His196 plays an important role in desolvation of the active site rather than stabilizing the transition state of the adenylation reaction. In addition, comparison of the new structure with a previously determined OSB-AMP-bound structure of the same enzyme allows us to propose a release mechanism of the C domain in its alteration to form the thioesterification conformation. These findings allow us to better understand the domain alternation catalytic mechanism of MenE as well as many other adenylating enzymes.

Structure of the adenylation domain Thr1 involved in the biosynthesis of 4‐chlorothreonine in Streptomyces sp. OH‐5093—protein flexibility and molecular bases of substrate specificity

“…Residues 170–171, within the P‐loop, are missing in the structure of this monomer. (B) Secondary structure and sequence comparison of Thr1, with PheA , BcDltA and bs MenE . The core motifs are boxed and labeled.…”

“…Comparison of the structure of Thr1 with other adenylation domains. (A) Overall superposition of the structures of substrates‐bound Thr1 (monomer A, in cyan) with product‐bound Thr1 (monomer B, in light green), bs MenE in complex with the product OSB ‐ AMP (pdb: , in orange) and BcDltA bound to the product d ‐Ala‐ AMP (pdb: , in light pink). The structures are superposed using the N‐subdomain as frame of reference, the rotation of the C‐subdomains around Asp423 at the beginning of a β‐hairpin occurring upon substrate adenylation in Thr1 is highlighted by arrows.…”

“…The e-amino group of Lys515 contributes to the stabilization of ATP by forming hydrogen bonds with O4 0 ribose ring oxygen (3.50 A) and with O5 0 ribose phosphoester oxygen (3.14 A). Moreover, this lysine is found at 2.59 A from the L-threonine carboxyl group, in , BcDltA [35] and bsMenE [36]. The core motifs are boxed and labeled.…”

Structures of the N‐Terminal Domain of PqsA in Complex with Anthraniloyl‐ and 6‐Fluoroanthraniloyl‐AMP: Substrate Activation in Pseudomonas Quinolone Signal (PQS) Biosynthesis

“…It has been demonstrated for several members of the ANL superfamily that the C‐terminal domain adopts different conformational states during the catalytic cycle through a mechanism called “domain alternation”, with a rotation of up to 140° around a conserved hinge residue . This flexibility is also reflected in elevated B ‐factors and disordered parts for the C‐terminal domain, which can be seen in the crystal structures of the acetoacetyl‐CoA ligase Sl Acs, the anthranilate‐CoA ligase AuaEII, or the o ‐succinylbenzoyl‐CoA ligase MenE, for example …”

“…[28][29][30][31][32][33] This flexibility is also reflected in elevated B-factors andd isordered parts for the C-terminal domain, which can be seen in the crystal structures of the acetoacetyl-CoA ligase SlAcs, the anthranilate-CoA ligase AuaEII, or the osuccinylbenzoyl-CoA ligase MenE, for example. [54][55][56] Because this mobility and flexibility might impede crystallization, we decided to use aP qsA construct lacking the whole Cterminald omain (PqsA NTD ), ending in the hinge residue (D399, Figure1Aand Figure S1 in the Supporting Information). Similar truncations have already been successfully employed in the crystallization of other ANL enzymes.…”