Molecular Dynamics Simulations of BcZBP, A Deacetylase from Bacillus cereus: Active Site Loops Determine Substrate Accessibility and Specificity

Abstract

BcZBP is an LmbE-like, homohexameric, zinc-dependent deacetylase from the opportunistic pathogen Bacillus cereus with three, thus far uncharacterized, homologues in B. anthracis. Although its specific substrate is still unknown, the enzyme has been shown to preferentially deacetylate N-acetylglucosamine and diacetylchitobiose via an active site based on a zinc-binding motif of the type HXDDXnH. In the crystal structure, the active site is located at a deep and partially blocked cleft formed at the interface between monomers related by the molecular 3-fold axis, although the major, in structural terms, building block of the enzyme is not the trimer, but the intertwined dimer. Here, we report results from a 50 ns molecular dynamics simulation of BcZBP in explicit solvent with full electrostatics and show that (i) the view of the intertwined dimer as the major structural and functional building block of this class of hexameric enzymes is possibly an oversimplification of the rather complex dynamics observed in the simulation, (ii) the most mobile (with respect to their atomic fluctuations) parts of the structure coincide with three surface loops surrounding the active site, and (iii) these mobile loops define the active site's accessibility, and may be implicated in the determination of the enzyme's specificity.

Characterization of the N-Acetyl-α-d-glucosaminyl l-Malate Synthase and Deacetylase Functions for Bacillithiol Biosynthesis in Bacillus anthracis,

“…These include a 10-residue insert I1 (relative to Bc ZBP) after Ser45, two small inserts (1-2 residues) following Bc ZBP Lys82 and Asp108, and two deletions D1 and D2 of 4-6 residues following Ile129 and Phe205, respectively. The I1 insert falls within the hypermobile Bc ZBP active-site loop L 46 , and the D1 deletion falls within the L 135 loop identified in the molecular dynamics study (24). Fadouloglou et al have implicated these loops (plus loop L 185 ) in active-site accessibility and substrate specificity.…”

“…Secondary structure assignments correspond to Bc ZBP; red boxes represent conserved residues, and yellow boxes represent conservative substitutions. Active-site loops L 46 , L 135 , and L 185 as defined for Bc ZBP (24) are indicated, as are the insert I1 and deletions D1 and D2 described in the text. Green and blue triangles represent ligands to the Bc ZBP active-site Zn 2+ and the proposed acid-base catalyst (Asp14) and charge-relay dyad (His110/Asp112), respectively.…”

LmbE proteins from Bacillus cereus are de‐N‐acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis

“…Based on the crystal structure of BC1534 [10] and a molecular dynamics study [20], we proposed that the rim and the loops surrounding the active site tunnel could play a significant role in the substrate specificity of the enzyme. We experimentally tested this hypothesis by mutating R140, K207 and A42, three residues located on the rim of the active site tunnel.…”

“…BC1534 is a hexamer (Fig. 3A) that may be considered as a trimer of dimers [10,20]. Dimerization is mainly established via exchange of two short β‐strands between the monomers (β8‐strand in Fig.…”

Cross Dimerization of Amyloid-β and αSynuclein Proteins in Aqueous Environment: A Molecular Dynamics Simulations Study

“…PCA has been widely recognized as a reliable starting point to identify important modes of interacting systems produced by MD simulations [45], [59]–[61]. The heavily populated clusters are identified by analysing the distribution of first three principal components using an rmsd cutoff of 2.4 Å.…”