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Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C--C ring closure reactions as opposed to thioester hydrolysis or O--C/N-C macrocyclization observed in previously reported TE structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B1, a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 [Angstrom] crystal structure displays an a/[beta]-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C-C cyclization. The biochemical studies, the 1.7 [Angstrom] crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C-C bond-forming class of TEs. biosynthesis | fungal metabolism | mutagenesis doi/10.1073/pnas.0913531107

Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona- and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-O-acetylesterase domains almost identical to corresponding domains in orthomyxo- and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine-Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptor-binding site seems to have been designed to use 9-mono- and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins. glycobiology | influenza | nidovirus | sialate-O-acetylesterase | X-ray crystallography

A study employs the X-ray crystallography technique to analyze the high-resolution crystal structure of a plant carboxylesterase, named Actinidia eriantha (AeCXEI), which is found to be closely related to the hormone-sensitive lipase subgroup structurally. The analysis reveals that the compound binds with paraoxon very easily forming a complex without inducing any change in the structure of the enzyme and hence helps in studying the role of carboxylesterases in plants.

Two chimeric enzymes, FLX and FLXLC, are constructed and successfully overproduced in Aspergillus niger. The results have shown that the fusion of naturally free cell wall hydrolases and an A. niger-derived carbohydrate-binding module (CBM) onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.

A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR were developed to test the ability of wild-type mutant forms of Phosphotriesterase (PTE) enzyme to hydrolyze these agents. Mutant forms of PTE identified with significantly enhanced as well as relaxed or reversed, stereoselectivity were observed to accurately predict a number of variants which exhibited remarkablly improved kinetic constants for the catalytic hydrolysis of the more toxic [S.sub.P] enantiomers.

A study was conducted to demonstrate the binding of C1 to enzymatically modified low-density lipoprotein (E-LDL) particles which involves recognition by the C1q globular domain of the unesterified fatty acids generated by cholesterol esterase. The direct evidence for the binding between C1q and E-LDL through its globular domains have potential implciations for antherogenesis study research.

X-ray structure crystallization of the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) was carried out to gain insight into the substitution of L29P which creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. The molecular modeling proposed that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds [epsi]-caprolactone more tightly.

The molecular dynamics and docking study of peptide dendrimers is described for the example of dendrimers catalyzing the hydrolysis of acyloxy pyrene trisulfonates in aqueous buffer. The studies have provided insights into the molecular dynamics of peptide dendrimers that are significant for other conformationally flexible dendrimers.

The purification, cloning and crystal structure characterization of Dr0930 enzyme, a member of the amidohydrolase superfamily in Deinococcus radiodurans is described. The phosphotriesterase activity of the intensely purple purified protein at high concentration which contains a mixture of zinc and iron is found to originate from an ancestral parent enzyme possessing lactonase activity.

The study results of an extensive human thioesterase superfamily member 2 (hTHEM2) substrate screens, the structural characterization of hTHEM2 complexed with the inert substrate analogue undecan-2-one-CoA and the kinetic analysis of active site mutants is presented. The results obtained for the structure-function analysis of hTHEM2 offer insight into the physiological role of hTHEM2, which involves catalysis of the hydrolysis of cytosolic medium-to-long-chain acyl-CoA thioesters.

The role of hydrogen bonding interactions in the substrate specificity of formation of an acyl-enzyme intermediate for the thioesterase (TE) from the 6-deoxyerythronolide B biosynthetic pathway is examined. The studies have shown that hydrophobic interactions between the binding cavity and substrate has driven substrate specificity, as observed in many protein-carbohydrate recognition events.

The biochemical characterization and cystallographic analysis of HI0827 protein isolated from Haemophilus influenza Rd KW20 was carried out to examine the structural variations within the acyl-CoA thioesterase family. The findings provided insight into a new structural class of hexameric hot dog fold enzyme, acyl-coenzyme A (acyl-CoA) thioesterases.

A study first validated a computational model of GNL7A1 by comparing the docked cocaine transition state analogue with the crystal structure and docked a tetrahedral approximation of the Ach transition state into the crystal structure of 7A1 Fab' bound to a cocaine transition state analogue to examine if the antibody would similarly stabilize the transition state for Ach hydrolysis. Results reveal that Ach to binds at the same site on the catalytic antibody and in a very similar conformation as cocaine.