Computational Insights into Amide Bond Formation Catalyzed by the Condensation Domain of Nonribosomal Peptide Synthetases

Abstract

Nonribosomal peptide synthetases (NRPSs) are important enzymes that synthesize an array of nongenetically encoded peptides. The latter have diverse physicochemical properties and roles. NRPSs are modular enzymes in which, for example, the condensation (C-) domain catalyzes the formation of amide bonds. The NRPS tyrocidine synthetase from Brevibacillus brevis is responsible for synthesizing the cyclic-peptide antibiotic tyrocidine. The first step is formation of an amide bond between a proline and phenylalanine which is catalyzed by a C-domain. In this study, a multiscale computational approach (molecular dynamics and QM/MM) has been used to investigate substrate binding and catalytic mechanism of the C-domain of tyrocidine synthetase. Overall, the mechanism is found to proceed through three exergonic steps in which an active site Histidine, His222, acts as a base and acid. First, His222 acts as a base to facilitate nucleophilic attack of the prolyl nitrogen at the phenylalanyl's carbonyl carbon. This is also the rate-limiting step with a free energy barrier of 38.8 kJ mol^-1. The second step is collapse of the resulting tetrahedral intermediate with cleavage of the S-C bond between the phenylalanyl and its Ppant arm, along with formation of the above amide bond. Meanwhile, the now protonated His222 imidazole has rotated toward the newly formed thiolate of the Ppant arm. In the final step, His222 acts as an acid, protonating the thiolate and regenerating a neutral His222. The overall mechanism is found to be exergonic with the final product complex being 46.3 kJ mol^-1 lower in energy than the initial reactant complex.

Scheme 1. Proposed Mechanism for the Amide Bond Formation between a Proline and Phenylalanine as Catalyzed by the Condensation Domain of the NRPS Tyrocidine Synthetase (RC = Reactant Complex; I = Intermediate; PC = Product Complex)

Figure 1

Illustration of the positioning, obtained by Docking, of the prolyl-loaded acceptor (^ASubs.) and phenylalanyl-loaded donor (^DSubs.) substrates within the active site of the NRPS tyrocidine synthetase.

Figure 2

(A) Plot of the rmsd of the active site residues His221, His222, Asp226, and Gly227 obtained over the course of the MD simulation and (B) snapshots taken from cluster analysis of the MD simulation, of the QM region residues and substrates.

Figure 3

Overlaid snapshots from the most populated cluster obtained from rmsd analysis of His222 in the MD simulation.

Figure 4

Optimized geometries (see Computational Methods) of the reactant and product complexes, intermediates, and transition structures obtained for tyrocidine synthetase’s catalyzed amide bond formation between the prolyl and phenylalanyl of ^ASubs. and ^DSubs. For clarity, only selected bond lengths (Å) and angles (°), and key components of the QM region are shown. Full structures are provided in Tables S1–S7.

Figure 5

Relative free energy (kJ mol^–1) surface obtained (see Computational Methods) for tyrocidine synthetase’s catalyzed amide bond formation between the prolyl and phenylalanyl moieties of ^ASubs. and ^DSubs.