title: 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 phenylacetyl-CoA 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.
authors.affiliation: From the School of Biomedical Sciences.
authors.forename: Yogesh B
authors.initials: YB
authors.lastname: Khandokar
authors.affiliation: From the School of Biomedical Sciences.
authors.forename: Parul
authors.initials: P
authors.lastname: Srivastava
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Boorooma Street, Wagga Wagga, New South Wales 2678 and.
authors.forename: Subir
authors.initials: S
authors.lastname: Sarker
authors.affiliation: From the School of Biomedical Sciences.
authors.forename: Crystall M D
authors.initials: CMD
authors.lastname: Swarbrick
authors.affiliation: the Australian Synchrotron, Blackburn Rd., Clayton, Victoria 3168, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Aragao
authors.affiliation: the Australian Synchrotron, Blackburn Rd., Clayton, Victoria 3168, Australia.
authors.forename: Nathan
authors.initials: N
authors.lastname: Cowieson
authors.affiliation: From the School of Biomedical Sciences,. Electronic address: jforwood@csu.edu.au.
authors.forename: Jade K
authors.initials: JK
authors.lastname: Forwood
mesh_terms: D000105:Acetyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000105:Acetyl Coenzyme A; C037816:phenylacetyl-coenzyme A; D013869:Thiolester Hydrolases; D003065:Coenzyme A
keywords: Streptococcus pneumoniae; crystal structure; fatty acyl-CoA; phenyl acetic acid; protein crystallization; protein purification; small-angle x-ray scattering (SAXS); thioesterase; x-ray crystallography