Mycobacteria Encode Active and Inactive Classes of TesB Fatty-Acyl CoA Thioesterases Revealed through Structural and Functional Analysis

Abstract

Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.

Thioesterase enzyme families: Functions, structures, and mechanisms

“…Within TesB TEs, this mutation appears to be unique to Mycobacterium species. 32 In TE6, Mus musculus Acot7 N-terminal domain (2V1O) and C-terminal domain (2Q2B) catalytic residues are reported as Asn24 and Asp213, respectively. 116 The structures for human Acot12 (3B7K, 4MOB, 4MOC) and M. musculus Acot7 (4ZV3, 6VFY) contain both N and Cterminal domains.…”

“…29 Structural knowledge about how enzymes perform thioester hydrolysis has increased; an insightful, recent review describes TE structures, with a particularly useful and clear connection of catalytic residues with enzyme topology. 30 Since we first classified TEs into families, new TE structures have been resolved, as examples: (a) in TE4, the TesB enzyme in Yersinia pestis was crystallized 31 ; as were (b) the TesB enzymes in mycobacteria 32 ; (c) in TE6, the human ACOT12 enzyme structure was obtained 33 ; (d) in TE11, the tertiary structure of the TE involved with azinomycin biosynthesis was determined 34 ;…”

Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

“…Purified Mycobacterium avium Ma TesB* has been shown to hydrolyze octanoyl-CoA and generate octanoic acid in vitro 43 . We neglected the octanoic acid synthesis activities by endogenous E. coli acyl-CoA thioesterase since Ec TesB generally has activities toward longer chain acyl-CoA (>C10) 44 and Ec YciA has activities toward shorter chain acyl-CoA (<C8) 45,46 .…”

Structure, function, and regulation of thioesterases

“…marinum (PDB 3U0A; [21]), M. avium subsp. paratuberculosis (PDB 4R9Z; [22]) and eukaryotes Saccharomyces cerevisiae (PDB 1TBU; [23]). Both prokaryotes and eukaryotes encode fused double hotdog domains (Figure 3), with a topology of 1-1-2-2-3-4-5-6-3-7-8-4-9-10-11-12, where the hotdog domain boundary is indicated by italics (Figure 4).…”

“…Figure22. TE17 family members possess a single / hydrolase fold which associated to form a homodimer.…”