Binding and thermodynamic properties of a carbohydrate binding module (CBM) and a glycoside hydrolase family 44 xyloglucanase/endoglucanase catalytic domain (CD) fromRuminococcus flavefaciens, both when separate and when linked to each other, have been quantified when binding various β-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.

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 acylACP 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 acylACP 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 acylACP 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.

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.

Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bonds on hybrid Quantum Mechanics::Molecular Mechanics energy surfaces. Eight such maps were constructed, including one for α-maltose and three for different forms of methyl α-acarviosinide to provide energies for twisting of α-(1,4) glycosidic bonds. Maps were also made for β-thiocellobiose and for three β-cellobiose conformers having different glycon ring shapes to model distortions of β-(1,4) glycosidic bonds. Different GH families twist scissile glycosidic bonds differently, increasing their potential energies from 0.5 to 9.5 kcal/mol. In general, the direction of twisting of the glycosidic bond away from the conformation of lowest intramolecular energy correlates with the position (syn or anti) of the proton donor with respect to the glycon’s ring oxygen atom. This correlation suggests that glycosidic bond distortion is important for the optimal orientation of one of the glycosidic oxygen lone pairs toward the enzyme’s proton donor.

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.

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.

Golgi α-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan₅GlcNAc₂ as part of the N-linked glycosylation pathway. To elucidate the molecular and electronic details of the reaction mechanism, in particular the conformation of the substrate at the transition state, we performed quantum mechanics/molecular mechanics metadynamics simulations of the glycosylation reaction catalyzed by GMII. The calculated free energy of activation for mannosyl glycosylation (23 kcal/mol) agrees very well with experiments, as does the conformation of the glycon mannosyl ring in the product of the glycosylation reaction (the covalent intermediate). In addition, we provide insight into the electronic aspects of the molecular mechanism that were not previously available. We show that the substrate adopts an ^OS₂/B_2,5 conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a D_NA_N reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an ^OS₂/B_2,5 → B_2,5 [TS] → ¹S₅ itinerary, agreeing with an earlier proposal based on comparing α- and β-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O2′−H_O2′ bond when the substrate acquires OCI character, relieving the electron deficiency of the OCI-like species. Our results can be used to explain the potency of recently formulated GMII anticancer inhibitors, and they are potentially relevant in deriving new inhibitors.

α-(1→2)-Mannosidase I from the endoplasmic reticulum (ERManI), a Family 47 glycoside hydrolase, is a key enzyme in the N-glycan synthesis pathway. Catalytic-domain crystal structures of yeast and human ERMan1s have been determined, the former with a hydrolytic product and the latter without ligands, with the inhibitors 1-deoxymannojirimycin and kifunensine, and with a thiodisaccharide substrate analog. Both inhibitors were bound at the base of the funnel-shaped active site as the unusual ¹C₄ conformer, while the substrate analog glycon is a ³S₁ conformer. In the current study, AutoDock was used to dock α-d-mannopyranosyl-(1→2)-α-d-mannopyranose with its glycon in chair (¹C₄, ⁴C₁), half-chair (³H₂, ³H₄, ⁴H₃), skew-boat (^OS₂, ³S₁, ⁵S₁), boat (^2,5B, ^3,OB, B_1,4, B_2,5), and envelope (³E, ⁴E, E₃, E₄) conformations into the yeast ERManI active site. Both docked energies and forces on docked ligand atoms were calculated to determine how the ligand distorts to the transition state. From these, we can conclude that (1) both ¹C₄ and^OS₂ can be the starting conformers; (2) the most likely binding pathway is¹C₄ → ³H₂ → ^OS₂ → ^3,OB → ³S₁ → ³E; (3) the transition state is likely to be close to a ³E conformation.

The ThYme (Thioester-active enzYme;http://www.enzyme.cbirc.iastate.edu) database has been constructed to bring together amino acid sequences and 3D (tertiary) structures of all the enzymes constituting the fatty acid synthesis and polyketide synthesis cycles. These enzymes are active on thioester-containing substrates, specifically those that are parts of the acyl-CoA synthase, acyl-CoA carboxylase, acyl transferase, ketoacyl synthase, ketoacyl reductase, hydroxyacyl dehydratase, enoyl reductase and thioesterase enzyme groups. These groups have been classified into families, members of which are similar in sequences, tertiary structures and catalytic mechanisms, implying common protein ancestry. ThYme is continually updated as sequences and tertiary structures become available.

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli.The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-Å resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and β-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55°C and an optimal pH of 5.0.