Structural mechanism of regioselectivity in an unusual bacterial acyl-CoA dehydrogenase

Abstract

Terminal alkenes are easily derivatized, making them desirable functional group targets for polyketide synthase (PKS) engineering. However, they are rarely encountered in natural PKS systems. One mechanism for terminal alkene formation in PKSs is through the activity of an acyl-CoA dehydrogenase (ACAD). Herein, we use biochemical and structural analysis to understand the mechanism of terminal alkene formation catalyzed by an γ,ẟ-ACAD from the biosynthesis of the polyketide natural product FK506, TcsD. While TcsD is homologous to canonical α,β-ACADs, it acts regioselectively at the γ,ẟposition and only on α,β-unsaturated substrates. Furthermore, this regioselectivity is controlled by a combination of bulky residues in the active site and a lateral shift in the positioning of the FAD cofactor within the enzyme. Substrate modeling suggests that TcsD utilizes a novel set of hydrogen bond donors for substrate activation and positioning, preventing dehydrogenation at the α,β position of substrates.From the structural and biochemical characterization of TcsD, key residues that contribute to regioselectivity and are unique to the protein family were determined and used to identify other putative γ,ẟ-ACADs that belong to diverse natural product biosynthetic gene clusters. These predictions are supported by the demonstration that a phylogenetically distant homolog of TcsD also regioselectively oxidizes α,β-unsaturated substrates. This work exemplifies a powerful approach to understand unique enzymatic reactions and will facilitate future enzyme discovery, inform enzyme engineering, and aid natural product characterization efforts.

Investigation of Indigoidine Synthetase Reveals a Conserved Active-Site Base Residue of Nonribosomal Peptide Synthetase Oxidases

“…Notably, we observed the Ppant ejection ion, a signature fragment ion of Ppant generated from CID, in tandem MS analysis ( Figure S3d). 25,26 Because the Ppant is acylated in thiotemplate biosynthesis, any changes in acylation can be tracked by monitoring changes in the molecular weight corresponding to the ejected Ppant ions from the PCP derived peptides, which can be first identified by the unchanged peptide fragment ions.…”

Functional Studies and Revision of the NFAT-133/TM-123 Biosynthetic Pathway in Streptomyces pactum

“…Examples of such enzymes include TcsD of the FK506 pathway in Streptomyces tsukubaensis NRRL 18488, which regioselectively dehydrogenates α,β-unsaturated pentenoyl-ACP at the γ,δ-position. 10 In such a scenario, the assembly line should start from C-1 and end at C-14 (for NFAT-133) or C-17 (for TM-123). Thus, NFAT-133 may be formed from a nascent heptaketide that is released in programmed fashion from the assembly line followed by decarboxylation.…”

“…The identification of several NFAT-133 analogues with a terminal olefin suggests that the pathway may involve online terminal dehydrogenation similar to that shown in FK506 biosynthesis. 10 The key enzyme that supports this notion is NftN, which has high amino acid identity (62%) to the acyl-CoA dehydrogenase TscD from S. tsukubaensis NRRL 18488. 10 Therefore, NftN may be responsible for the formation of the terminal olefin shown in TM-125, TM-126, TM-131, and TM-132.…”

“…10 The key enzyme that supports this notion is NftN, which has high amino acid identity (62%) to the acyl-CoA dehydrogenase TscD from S. tsukubaensis NRRL 18488. 10 Therefore, NftN may be responsible for the formation of the terminal olefin shown in TM-125, TM-126, TM-131, and TM-132. To test this hypothesis, the nftN gene was inactivated by in-frame deletion of the gene in the ΔptmTDQ strain.…”

Benzylmalonyl-CoA dehydrogenase, an enzyme involved in bacterial auxin degradation

“…They usually use an electron-transfer flavoprotein (ETF) as electron acceptor, which transfers the redox equivalents further to ubi- or menaquinones via ETF:quinone oxidoreductases (Ghisla and Thorpe 2004 ; Frerman 1987 ). Other members of the family are involved in β-condensation reactions as enoyl-CoA or enoyl-acyl carrier protein (ACP) reductases, using NAD(P)H as reductant (Ghisla and Thorpe 2004 ), or catalyze apparently unrelated reactions such as desulfination or γ,δ-double bond generation (Schürmann et al 2015 ; Blake-Hedges et al 2020 ). The function of IaaF in the proposed indoleacetate catabolic pathway is the oxidative decarboxylation of (2-aminobenzyl)malonyl-CoA to 2-aminocinnamoyl-CoA and CO 2 as shown in Fig.…”