A Reaction Path Study of the Catalysis and Inhibition of theBacillus anthracisCapD γ-Glutamyl Transpeptidase

Abstract

The CapD enzyme of Bacillus anthracis is a γ-glutamyl transpeptidase from the N-terminal nucleophile hydrolase superfamily that covalently anchors the poly-γ-D-glutamic acid (pDGA) capsule to the peptidoglycan. The capsule hinders phagocytosis of B. anthracis by host cells and is essential for virulence. The role CapD plays in capsule anchoring and remodeling makes the enzyme a promising target for anthrax medical countermeasures. Although the structure of CapD is known, and a covalent inhibitor, capsidin, has been identified, the mechanisms of CapD catalysis and inhibition are poorly understood. Here, we used a computational approach to map out the reaction steps involved in CapD catalysis and inhibition. We found that the rate-limiting step of either CapD catalysis or inhibition was a concerted asynchronous formation of the tetrahedral intermediate with a barrier of 22-23 kcal/mol. However, the mechanisms of these reactions differed for the two amides. The formation of the tetrahedral intermediate with pDGA was substrate-assisted with two proton transfers. In contrast, capsidin formed the tetrahedral intermediate in a conventional way with one proton transfer. Interestingly, capsidin coupled a conformational change in the catalytic residue of the tetrahedral intermediate to stretching of the scissile amide bond. Furthermore, capsidin took advantage of iminol-amide tautomerism of its diacetamide moiety to convert the tetrahedral intermediate to the acetylated CapD. As evidence of the promiscuous nature of CapD, the enzyme cleaved the amide bond of capsidin by attacking it on the opposite side compared to pDGA.

N-Acylethanolamine Acid Amidase (NAAA): Mechanism of Palmitoylethanolamide Hydrolysis Revealed by Mechanistic Simulations

“…Prior theoretical investigations on the catalytic mechanism of Ntn-hydrolases in the presence of substrates include penicillin acylase (PA), aspartylglucosaminidase (AGA), γ-glutamyl transpeptidase (GGT), and conjugated bile acid hydrolase (CBAH) . PA is a serine hydrolase, and AGA and GGT are threonine hydrolases, while CBAH is a cysteine hydrolase belonging to the same enzyme superfamily of NAAA .…”

β-Aminoalcohols as Potential Reactivators of Aged Sarin-/Soman-Inhibited Acetylcholinesterase

“…Our earlier calculations demonstrated that the nucleophilic OγH group of the β‐aminoalcohol moiety from the N‐terminal catalytic threonine of CapD is deprotonated during the nucleophilic attack on its substrate, transferring its proton to the originally neutral N‐terminal amino group . Unlike oximes, the β‐aminoalcohol nucleophile is neutral at the beginning of the nucleophilic attack but develops a negative charge during an intramolecular proton transfer.…”

“…To test whether the designed molecules could reactivate aged AChE, we used a multi‐tiered computational chemistry approach comprising 1 ) docking of a molecule into the active site of the aged AChE adduct, 2 ) molecular dynamic simulations of the complex in solution, and 3 ) hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations to assess the steps and energetics of aged AChE reactivation. Details of the approach are provided in the Supporting Information.…”

Treatment of experimental anthrax with pegylated circularly permuted capsule depolymerase

“…CapD is a γ-glutamyltransferase (GGT) that catalyzes endolytic cleavage of PDGA and degrades the capsular material to low–molecular weight multimers by catalyzing the transfer of a γ-glutamyl group to either water or an amino acid acceptor ( 4 , 24 , 25 ). The polymer of D-Glu is not present in humans.…”

“…CapD must autocatalytically cleave itself internally to produce a free N-terminal Thr 352 . Thr 352 acts as the general acid, general base, and nucleophile in the reaction ( 24 , 25 ). The internal cleavage produces two fragments (residues 1 to 351 and 352 to 528), and the activity of GGT enzymes is limited by the efficiency of autoproteolysis.…”