Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA

Abstract

Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.

Keywords: 2-hydroxyacyl-CoA synthase; Actinomycetospora; carbonyl compounds; formate assimilation; glycolyl-CoA; mono-carbon extension; oxalyl-CoA decarboxylase; x-ray diffraction.

Figure 1

Schematic comparison of HACL and OXC-catalyzed C-C cleavage reaction.

Figure 2

AcHACL enzyme structures trapped in different ligand-bound states.A, the tetrameric AcHACL enzyme architecture can be described as a dimer of dimers. B, the top view of one dimer shows that the active sites are located between two protein chains. C–F, zoom into the active site of (C) the substrate-bound crystal structure, which was obtained in the presence of the inactive cofactor dzThDP, (D) the first intermediate bound modeled state in which cofactor ThDP is covalently bound to 2-HIB-CoA, (E) the second intermediate bound crystal structure and (F) the product-bound enzyme structure. The ligands are shown in light blue. The ligand interacting enzyme residues are colored in green and yellow according to the protein chain from which they originate. Omit electron density maps are shown for the crystal structures at 2.5 σ level. Formyl-CoA is depicted in dim colors because the formyl-cysteamine-β-alanyl residue is not well defined in the electron density. Hydrogen bonds between the ligands and the protein are shown as black dashed lines.

Figure 3

AcHACL mutant enzyme structures. Zoom into the active site of (A) the substrate-bound crystal structure of the AcHACL mutant E493Q, (B) the substrate-bound crystal structure of the enzyme mutant E493A, and (C) the first intermediate bound modeled state of the AcHACL mutant E493A. The color scheme is the same as in Figure 2. Omit electron density maps are shown for the crystal structures at 2.5 σ level.

Figure 4

Proposed catalytic cycle for the WT AcHACL-catalyzed reaction.

Figure 5

Active site architecture of AcHACL and enzyme homologs.A, the size of the active site of AcHACL is restricted by the E493-containing α-helix (light green) and the C-terminal lid domain (dark green). The enzyme residues interacting with the 2-HIB residue are colored in green and yellow according to the protein chain from which they originate. The modeled first intermediate is shown in light blue. Hydrogen bonds between the 2-HIB residue and the protein are shown as dashed black lines. B, superposition of the crystal structures of AcHACL (green) and BsALS (beige, PDB ID: 4RJI) with the AlphaFold model of HsHACL2 (violet, AF-A1L0T0-F1) shows that the active site α-helix is in a closed conformation for all three structures. Residue E493 forms an interaction with the 2-hydroxyl group of the first intermediate (light blue) in AcHACL, whereas HsHACL2 and BsALS have an isoleucine and valine residue, respectively, at this position that prevents H-bonding interactions. However, in HsHACL2 and several related enzymes, a glutamic acid is present three residues later (one helix turn) in the sequence within the α-helix (Fig. S3), which is possibly involved in substrate interactions. In BsALS, the enzymatic mechanism is different, but the glutamine residue Q487 is located next to the AcHACL intermediate within the superimposed structures, thereby illustrating a possible interaction mechanism for HACL enzymes that is highlighted as dashed red line. Values refer to atomic distance in Å. C–E, zoom into the active site of the superposition of the second intermediate bound AcHACL crystal structure with the crystal structures of (C) BsALS in complex with a 2-lactyl bound ThDP intermediate (PDB ID: 4RJK), (D) RuHACL in complex with the cofactor analog TzDP (PDB ID: 6XN8) and (E) OfOXC with covalently bound second intermediate (PDB ID: 2JI7). The ligands and ligand interacting amino acids are colored in beige, orange, and brown, respectively, for the structural homologs.