Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn and Asp as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase.

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...

Precise identification of correct exon–intron boundaries is a prerequisite to analyze the location and structure of genes. The existing framework for genomic signals, delineating exon and introns in a genomic segment, seems insufficient, predominantly due to poor sequence consensus as well as limitations of training on available experimental data sets. We present here a novel concept for characterizing exon–intron boundaries in genomic segments on the basis of structural and energetic properties. We analyzed boundary junctions on both sides of all the exons (3 28 368) of protein coding genes from human genome (GENCODE database) using 28 structural and three energy parameters. Study of sequence conservation at these sites shows very poor consensus. It is observed that DNA adopts a unique structural and energy state at the boundary junctions. Also, signals are somewhat different for housekeeping and tissue specific genes. Clustering of 31 parameters into four derived vectors gives some additional insights into the physical mechanisms involved in this biological process. Sites of structural and energy signals correlate well to the positions playing important roles in pre-mRNA splicing.

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.

Lack of a consistent PM (particulate matter smaller than 10 μm) database at high spatial resolution hinders in assessing the environmental impact of PM in India. Here we propose an alternate approach to estimate the PM database. Aerosol extinction coefficients at the surface are calculated from midvisible aerosol optical depth from MERRA-2 reanalysis data using characteristics vertical profiles from CALIOP and then are converted to PM mass using aerosol property information and microphysical data. The retrieved PM are bias-corrected and evaluated ( R = 0.85) against coincident ground-based data maintained under the Central Pollution Control Board network. PM exposure exceeds Indian annual air quality standard in 72.3% districts. Transition in PM exposure from the monsoon (Jun-Sep) to postmonsoon season (Oct-Nov) translates to 1-2% higher all-cause mortality risk over the polluted Indo-Gangetic Basin (IGB). Mortality risk increases in the central to eastern IGB and central India and reduces in Delhi national capital region in the winter (Dec-Feb) relative to the postmonsoon season. Mortality risk decreases by 0.5-1.8% in most parts of India in the premonsoon season (Mar-May). Our results quantify the vulnerability in terms of seasonal transition in all-cause mortality risks due to PM exposure at district level for the first time in India.