Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism

Abstract

PaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn 37 , Asp 52 , and Thr 68 are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N terminus, and a loop region involving Tyr 38 -Tyr 39 . This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetylCoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family. Thioesterases (TEs)2 are a superfamily of enzymes responsible for the catalysis of thioester bonds between carbonyl and thiol groups. Due to their wide range of cellular functions, diverse substrate specificity, and structural differences, they have been classified into 23 families (1). TE families 1-13 hydrolyze thioester bonds between acyl moieties and CoA, TEs 14 -19 exhibit preference to acyl groups linked to ACP, TEs 20 -21 cleave thioester bonds between acyl groups and non-ACP proteins, and TEs 22-23 hydrolyze the glutathione and its derivatives from acyl groups. There are 5-folds within the TE superfamily, NagB, ␣/␤-hydrolase, flavodoxin-like, hotdog, and lactamase, with the hotdog and ␣/␤-hydrolase folds most prevalent (1).The thioesterase characterized in this study is a member of the TE13 family. To date, members of this family include PaaI and PaaD, and have been structurally resolved from Thermus thermophilus (Protein Data Bank (PDB) 1J1Y) (2) and Escherichia coli (PDB 2FS2) (3). Biochemical and structural characterization of PaaI from these organisms has established that the enzymes form a tetrameric hotdog fold, with catalytic specificity toward the hydrolysis of phenylacetyl-CoA (3). The arrangement of the hotdog folds in these enzymes has been described as a back to back configuration, such that the double hotdog domain are arranged with t...

Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase

“…Because the specificity of the enzyme has not been determined previously, we screened a range of substrates using an established 5,5Ј-dithiobis(nitrobenzoic acid) assay (26,27) to identify the substrate specificity. Substrates ranging in carbon chain length from two (C2) to 20 (C20) were screened, with the highest activity observed for acetyl-CoA (Fig.…”

A family 13 thioesterase isolated from an activated sludge metagenome: Insights into aromatic compounds metabolism

“…This zone can be found as a straight helix (as in 2FS2), a bent helix (as in 3F5O, 4ORD, 1VH9, 1VI8, 4K02, 5HMB, 1J1Y, and 1ZKI), a short helix followed by a loop (1Q4S), or an open segment that is involved in domain swapping (4ZRB). The position of the N‐terminus affects the conformation of the loop following the C‐terminus of the long helix (the hotdog) and the first beta turn of the “bun”, so it does not have a direct effect on the composition of the active site, but it can block access . In view of the similarity of C‐scores of the unbiased model and those derived from structures with the most common geometry for the N‐terminus of the TE hotdog fold, we carried out all further analysis with the unbiased model.…”

Structure, function, and regulation of thioesterases

“…Within related hotdog families several crystal structures have shown only half of the domains contain CoA, despite all domains containing equivalent active site configurations (PDBs: 4MOB, 4MOC [43], 3B7K, 4ZV3). Moreover, other single hotdog domain TE family members also exhibit CoA bound at only a fraction of the sites (PDBs 4R4U [17], 4ZRB [64], 5BYU, 5KL9). Substrate-induced structural rearrangements have been described in other thioesterase families [17,64], although the exact functional role for half-of-sites activity from prokaryotes through to eukaryotes remains to be established.…”

“…Moreover, other single hotdog domain TE family members also exhibit CoA bound at only a fraction of the sites (PDBs 4R4U [17], 4ZRB [64], 5BYU, 5KL9). Substrate-induced structural rearrangements have been described in other thioesterase families [17,64], although the exact functional role for half-of-sites activity from prokaryotes through to eukaryotes remains to be established. A half-of-sites reactivity has also been described more broadly, with a large number of enzymes including aldehyde J o u r n a l P r e -p r o o f dehydrogenase [65], thymidylate synthase [66], aspartate receptors [67] and bacterial adenylyltransferase [68], all displaying a half-of-sites activity.…”

“…The TE13 thioesterase family members are PaaI type thioesterases, named for their presence in the phenylacetic acid paa gene cluster, and exhibit activity against short and medium chain acyl-CoA, as well as several hydroxyphenylacetyl-CoA substrates. They exhibit a single hotdog fold, and representative structures have been solved from E. coli (PDB 1PSU and 2FS2; [114]), P. aeruginosa (PDB 1ZKI; [115]), S. pneumoniae (PDB 4I82, 4ZRF, 4ZRB, 4XY5 and 4XY6; [64]), S. mutans (PDB 3LBB and 3LBE; [116]), S. aureus (PDB 4M20, 5EP5; [117], 4YBV; [118]) and T. thermophilus (PDB 1J1Y, 1WLU, 1WLV, 1WM6, 1WN3; [119] and 2DSL; [120]). The topology of this family is arranged as 1-1-2-2-3-3-4-5-6 with residues important for catalysis including Asn, Asp and Thr (SpPaaI).…”