Glycoside hydrolases cleave the glycosidic linkage between two carbohydrate moieties. They are among the most efficient enzymes currently known. β-Xylosidases from glycoside hydrolase family 43 hydrolyze the nonreducing ends of xylooligomers using an inverting mechanism. Although the general mechanism and catalytic amino acid residues of β-xylosidases are known, the nature of the reaction’s transition state and the conformations adopted by the glycon xylopyranosyl ring along the reaction pathway are still elusive. In this work, the xylobiose hydrolysis reaction catalyzed by XynB3, a β-xylosidase produced byGeobacillus stearothermophilus T-6, was explicitly modeled using first-principles quantum mechanics/molecular mechanics Car−Parrinello metadynamics. We present the reaction’s free energy surface and its previously undetermined reaction pathway. The simulations also show that the glycon xylopyranosyl ring proceeds through a 2,5B-type transition state with significant oxacarbenium ion character. Disciplines Biochemical and Biomolecular Engineering | Biological Engineering | Chemical Engineering Comments Posted with permission from The Journal of Physical Chemistry B, 114, no. 46 (2010): 15389–15393, doi:10.1021/jp107886e. Copyright 2010 American Chemical Society. This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/cbe_pubs/13 Mechanism of Xylobiose Hydrolysis by GH43 -Xylosidase Ian J. Barker, Luis Petersen, and Peter J. Reilly* Department of Chemical and Biological Engineering, Iowa State UniVersity, Ames, Iowa 50011, United States ReceiVed: August 19, 2010; ReVised Manuscript ReceiVed: September 25, 2010 Glycoside hydrolases cleave the glycosidic linkage between two carbohydrate moieties. They are among the most efficient enzymes currently known. -Xylosidases from glycoside hydrolase family 43 hydrolyze the nonreducing ends of xylooligomers using an inverting mechanism. Although the general mechanism and catalytic amino acid residues of -xylosidases are known, the nature of the reaction’s transition state and the conformations adopted by the glycon xylopyranosyl ring along the reaction pathway are still elusive. In this work, the xylobiose hydrolysis reaction catalyzed by XynB3, a -xylosidase produced by Geobacillus stearothermophilus T-6, was explicitly modeled using first-principles quantum mechanics/molecular mechanics Car-Parrinello metadynamics. We present the reaction’s free energy surface and its previously undetermined reaction pathway. The simulations also show that the glycon xylopyranosyl ring proceeds through a 2,5B-type transition state with significant oxacarbenium ion character.

We classified the carboxylic ester hydrolases (CEHs) into families and clans by use of multiple sequence alignments, secondary structure analysis, and tertiary structure superpositions. Our work for the first time has fully established their systematic structural classification. Family members have similar primary, secondary, and tertiary structures, and their active sites and reaction mechanisms are conserved. Families may be gathered into clans by their having similar secondary and tertiary structures, even though primary structures of members of different families are not similar. CEHs were gathered from public databases by use of Basic Local Alignment Search Tool (BLAST) and divided into 91 families, with 36 families being grouped into five clans. Members of one clan have standard α/β-hydrolase folds, while those of other two clans have similar folds but with different sequences of their β-strands. The other two clans have members with six-bladed β-propeller and three-α-helix bundle tertiary structures. Those families not in clans have a large variety of structures or have no members with known structures. At the time of writing, the 91 families contained 321,830 primary structures and 1378 tertiary structures. From these data, we constructed an accessible database: CASTLE (CArboxylic eSTer hydroLasEs, http://www.castle.cbe.iastate.edu).

The carboxylic ester hydrolases (CEHs) are enzymes that hydrolyze an ester bond to form a carboxylic acid and an alcohol. They are one of the enzyme groups that are most explored industrially for their applications in the food, flavor, pharmaceutical, organic synthesis, and detergent industries. We classified CEHs into families and clans according to their amino acid sequences (primary structures) and three-dimensional structures (tertiary structures). Our work has established the systematic structural classification of the CEHs. Primary structures of family members are similar to each other, and their active sites and reaction mechanisms are conserved. The tertiary structures of members of each clan, which is composed of different families, remain very similar, although amino acid sequences of members of different families are not similar. CEHs were divided into 127 families by use of BLAST, with 67 families being grouped into seven clans. Multiple sequence alignment and tertiary structures superposition were used, and active sites and reaction mechanisms were analyzed. Python and Shell scripts were implemented to automate the process of comparing CEH primary and tertiary structures. A comprehensive database, CASTLE (CArboxylic eSTer hydroLasEs), may be constructed to provide the primary and tertiary structures of CEHs. This database would be available at www.castle.enzyme.iastate.edu and will be accessible to the entire biology community.

ABSTRACTAt present, 69 families of carbohydrate‐binding modules (CBMs) have been isolated by statistically significant differences in the amino acid sequences (primary structures) of their members, with most members of different families showing little if any homology. On the other hand, members of the same family have primary and tertiary (three‐dimensional) structures that can be computationally aligned, suggesting that they are descended from common protein ancestors. Members of the large majority of CBM families are β‐sandwiches. This raises the question of whether members of different families are descended from distant common ancestors, and therefore are members of the same tribe. We have attacked this problem by attempting to computationally superimpose tertiary structure representatives of each of the 53 CBM families that have members with known tertiary structures. When successful, we have aligned locations of secondary structure elements and determined root mean square deviations and percentages of similarity between adjacent amino acid residues in structures from similar families. Further criteria leading to tribal membership are amino acid chain lengths and bound ligands. These considerations have led us to assign 27 families to nine tribes. Eight of the tribes have members with β‐sandwich structures, while the ninth is composed of structures with β‐trefoils. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 203–214, 2015.

Automated docking allows rapid screening of protein-ligand interactions. A scoring function composed of a force field and linear weights can be used to compute a binding energy from a docked atom configuration. For different force fields or types of molecules, it may be necessary to train a custom scoring function. This chapter describes the data and methods one must consider in developing a custom scoring function for use with AutoDock.

Ketoacyl reductases (KRs), hydroxyacyl dehydratases (HDs), and enoyl reductases (ERs) are part of the fatty acid/polyketide synthesis cycle. They are known as acyl dehydrogenases, enoyl hydratases, and hydroxyacyl dehydrogenases, respectively, when catalyzing their reverse reactions. Earlier, we classified these enzymes into four KR, eight HD, and five ER families by statistical criteria. Members of all four KR families and three ER families have Rossmann folds, while five HD family members have HotDog folds. This suggests that those proteins with the same folds in different families may be distantly related, and therefore in clans, even though their amino acid sequences may not be homologous. We have now defined two clans containing three of the four KR families and two of the eight HD families, using manual and statistical tests. One of the ER families is related to the KR clan.

This article is a review of computational work over the last ∼15 years to elucidate the catalytic mechanisms of three major enzyme classes: glycoside hydrolases, carboxylic ester hydrolases, and thioesterases. The mechanisms of glycoside hydrolases that both invert and retain the configuration of the anomeric carbon atom of the labile glycosidic bond are covered, as is the twisting of the glycosidic bond exerted by members of different glycoside hydrolase families. The hydrolytic mechanisms and substrate binding of five different carboxylic ester hydrolase classes—acetylcholinesterases, butyrylcholinesterases, cocaine esterases, carboxylesterases, and triacylglycerol lipases—are addressed. Finally, the mechanism of members of a thioesterase family with HotDog tertiary structures is covered.

Thioesterases are enzymes that hydrolyze thioester bonds between a carbonyl group and a sulfur atom. They catalyze key steps in fatty acid biosynthesis and metabolism, as well as polyketide biosynthesis. The reaction molecular mechanism of most hotdog-fold acyl-CoA thioesterases remains unknown, but several hypotheses have been put forward in structural and biochemical investigations. The reaction of a human thioesterase (hTHEM2), representing a thioesterase family with a hotdog fold where a coenzyme A moiety is cleaved, was simulated by quantum mechanics/molecular mechanics metadynamics techniques to elucidate atomic and electronic details of its mechanism, its transition-state conformation, and the free energy landscape of the process. A single-displacement acid-base-like mechanism, in which a nucleophilic water molecule is activated by an aspartate residue acting as a base, was found, confirming previous experimental proposals. The results provide unambiguous evidence of the formation of a tetrahedral-like transition state. They also explain the roles of other conserved active-site residues during the reaction, especially that of a nearby histidine/serine pair that protonates the thioester sulfur atom, the participation of which could not be elucidated from mutation analyses alone.

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well.

Binding and thermodynamic properties of a carbohydrate binding module (CBM) and a glycoside hydrolase family 44 xyloglucanase/endoglucanase catalytic domain (CD) from Ruminococcus flavefaciens, both when separate and when linked to each other, have been quantified when binding various beta-1,4-linked glucans and xylans. The three constructs bind cellotetraose, cellopentaose, and cellohexaose with association constants that increase with chain length. The CBM does not bind xylotetraose, xylopentaose, or xylohexaose. The CD appears to bind carboxymethylcellulose (CMC) and xylan only weakly, while the CBM and the CD/CBM bind them much more strongly than they bind the cellooligosaccharides. CMC is bound to a much greater degree than is xylan. Association constants for the cellooligosaccharides are in the order CBM << CD < CD/CBM, while those on CMC and xylan are CD << CBM CD/CBM. A synergistic effect was observed for the association constants of cellopentaose and cellohexaose with the CD/CBM when compared to the CD and CBM alone. Binding of all ligands by all three constructs is energetically favorable, enthalpy-driven, and subject to enthalpy-entropy compensation.

We gathered primary and tertiary structures of acyl-CoA carboxylases from public databases, and established that members of their biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains occur in one family each and that members of their carboxyl transferase (CT) domains occur in two families. Protein families have members similar in primary and tertiary structure that probably have descended from the same protein ancestor. The BCCP domains complexed with biotin in acyl and acyl-CoA carboxylases transfer bicarbonate ions from BC domains to CT domains, enabling the latter to carboxylate acyl and acyl-CoA moieties. We separated the BCCP domains into four subfamilies based on more subtle primary structure differences. Members of different BCCP subfamilies often are produced by different types of organisms and are associated with different carboxylases.

Ketoacyl reductases (KRs), hydroxyacyl dehydratases (HDs) and enoyl reductases (ERs) are part of the fatty acid and polyketide synthesis cycles. Their reverse reactions, catalyzed by acyl dehydrogenases (equivalent to ERs), enoyl hydratases (equivalent to HDs) and hydroxyacyl dehydrogenases (equivalent to KRs), are part of fatty acid degradation by β-oxidation. These enzymes have been classified into families based on similarities in their primary and tertiary structures, and these families and their structures are included in the ThYme (Thioester-active enzYmes) database. Members of each family have strong sequence similarity and have essentially the same tertiary structure, mechanism and catalytic residues.

Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm ( Elaeis guineensis ), coconut ( Cocos nucifera ) and Cuphea viscosissima , organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli . Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Ketoacyl synthases (KSs) catalyze condensing reactions combining acyl-CoA or acyl-acyl carrier protein (acyl-ACP) with malonyl-CoA to form 3-ketoacyl-CoA or with malonyl-ACP to form 3-ketoacyl-ACP. In each case, the resulting acyl chain is two carbon atoms longer than before, and CO2 and either CoA or ACP are formed. KSs also join other activated molecules in the polyketide synthesis cycle. Our classification of KSs by their primary and tertiary structures instead of by their substrates and the reactions that they catalyze enhances insights into this enzyme group. KSs fall into five families separated by their characteristic primary structures, each having members with the same catalytic residues, mechanisms, and tertiary structures. KS1 members, overwhelmingly named 3-ketoacyl-ACP synthase III or its variants, are produced predominantly by bacteria. Members of KS2 are mainly produced by plants, and they are usually long-chain fatty acid elongases/condensing enzymes and 3-ketoacyl-CoA synthases. KS3, a very large family, is composed of bacterial and eukaryotic 3-ketoacyl-ACP synthases I and II, often found in multidomain fatty acid and polyketide synthases. Most of the chalcone synthases, stilbene synthases, and naringenin-chalcone synthases in KS4 are from eukaryota. KS5 members are all from eukaryota, most are produced by animals, and they are mainly fatty acid elongases. All families except KS3 are split into subfamilies whose members have statistically significant differences in their primary structures. KS1 through KS4 appear to be part of the same clan. KS sequences, tertiary structures, and family classifications are available on the continuously updated ThYme (Thioester-active enzYme) database.

Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose.