Peter J. Reilly

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

BackgroundAcyl-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.ResultsTo 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.ConclusionThese acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Modeling of aldopyranosyl ring puckering with MM3 (92)

Abstract The molecular mechanics algorithm MM3 was used to compute energy surfaces for aldopyranosyl rings having a full range of shapes. Energies were plotted against the ΦΘ puckering coordinates of Cremer and Pople. The 4 C 1 conformations of the model pyranosyl rings are dominant for both anomers of d -allose, d -galactose, d -glucose, d -mannose, and d -talose, as are the 4 C 1 conformations of β- d -altropyranose, β- d -gulopyranose, and β- d -idopyranose. α- d -Altropyranose is predicted to exist as an equilibrium of 1 C 4 and 4 C 1 , α- d -idopyranose as an equilibrium among O S 2 , 1 C 4 , and 4 C 1 , and α- d -gulopyranose is predominately 4 C 1 but has some contribution from 1 C 4 (18%) and O S 2 (9%). The calculated and measured hydrogen-hydrogen coupling constants agree well, although the energies for the β anomers in water are systematically low by an average of 0.4 kcal/mol. Because the errors in the predicted anomeric ratios are small and are similar for the eight hexoses, and because the only concession to the solvent was a dielectric constant of 3.0, specific solvent effects are apparently small.

Glucoamylase structural, functional, and evolutionary relationships

To correlate structural features with glucoamylase properties, a structure‐based multisequence alignment was constructed using information from catalytic and starch‐binding domain models. The catalytic domain is composed of three hydrophobic folding units, the most labile and least hydrophobic of them being missing in the most stable glucoamylase. The role of O‐glycosylation in stabilizing the most hydrophobic folding unit, the only one where thermostabilizing mutations with unchanged activity have been made, is described. Differences in both length and composition of interhelical loops are correlated with stability and selectivity characteristics. Two new glucoamylase subfamilies are defined by using homology criteria. Protein parsimony analysis suggests an ancient bacterial origin for the glucoamylase gene. Increases in length of the belt surrounding the active site, degree of O‐glycosylation, and length of the linker probably correspond to evolutionary steps that increase stability and secretion levels of Aspergillus‐related glucoamylases. Proteins 29:334–347, 1997.

Conformational analysis of the anomeric forms of sophorose, laminarabiose, and cellobiose using MM3

Relaxed-residue energy maps based on the MM3 force-field were computed for relative orientations of the pyranosyl rings of sophorose, laminarabiose, and cellobiose, respectively the (1----2)-beta-; (1----3)-beta-; and (1----4)-beta-linked D-glucosyl disaccharides. Sixteen starting conformations of the rotatable exocyclic side-groups were considered for each molecule. All of the energy surfaces have two intersecting low-energy troughs and illustrate the importance of exo-anomeric effects in determining disaccharide conformation. Local minima were found by relaxed minimization without restriction. The energy surfaces of these disaccharides are very similar to the energy surfaces of their corresponding 6-methyltetrahydropyran analogues. There is good agreement between disaccharide structures having minimal MM3 energy and those found by crystallography.

Thioesterases: A new perspective based on their primary and tertiary structures

Thioesterases (TEs) are classified into EC 3.1.2.1 through EC 3.1.2.27 based on their activities on different substrates, with many remaining unclassified (EC 3.1.2.–). Analysis of primary and tertiary structures of known TEs casts a new light on this enzyme group. We used strong primary sequence conservation based on experimentally proved proteins as the main criterion, followed by verification with tertiary structure superpositions, mechanisms, and catalytic residue positions, to accurately define TE families. At present, TEs fall into 23 families almost completely unrelated to each other by primary structure. It is assumed that all members of the same family have essentially the same tertiary structure; however, TEs in different families can have markedly different folds and mechanisms. Conversely, the latter sometimes have very similar tertiary structures and catalytic mechanisms despite being only slightly or not at all related by primary structure, indicating that they have common distant ancestors and can be grouped into clans. At present, four clans encompass 12 TE families. The new constantly updated ThYme (Thioester‐active enzYmes) database contains TE primary and tertiary structures, classified into families and clans that are different from those currently found in the literature or in other databases. We review all types of TEs, including those cleaving CoA, ACP, glutathione, and other protein molecules, and we discuss their structures, functions, and mechanisms.

Xylanases: structure and function.

A number of papers in this seminar are devoted to the production and characterization of cellulases. This is proper, since the use of cellulases is perhaps the most feasible method of converting cellulose to products that may be used for fuels and chemicals. However, all celluloses from hardwoods and grasses are associated with significant amounts of xylan, a hemicellulose possessing a β-l,4-linked xylose backbone, with branches containing xylose and other pentoses, hexoses, and uronic acids (Figure 1).

Conformational analysis of the anomeric forms of kojibiose, nigerose, and maltose using MM3

Energy surfaces were computed for relative orientations of the relaxed pyranosyl rings of the two anomeric forms of kojibiose, nigerose, and maltose, the (1----2)-alpha, (1----3)-alpha, and (1----4)-alpha-linked D-glucosyl disaccharides, respectively. Twenty-four combinations of starting conformations of the rotatable side-groups were considered for each disaccharide. Optimized structures were calculated using MM3 on a 20 degree grid spacing of the torsional angles about the glycosidic bonds. The energy surfaces of the six disaccharides were similar in many respects but differed in detail within the low-energy regions. The maps also illustrate the importance of the exo-anomeric effect and linkage type in determining the conformational flexibility of disaccharides. Torsional conformations of known crystal structures of maltosyl-containing molecules lie in a lower MM3 energy range than previously reported.

Purification and characterization of a xylobiose- and xylose-producing endo-xylanase from Aspergillus niger

Abstract A xylanase from a commercial Aspergillus niger pentoglycanase was purified to homogeneity by column chromatography on Ultrogel AcA 54, SP-Sephadex, Sephadex G-50, and SP-Sephadex. The enzyme hydrolyzed xylotriose slowly to xylose and xylobiose, and xylotetraose and higher xylo-oligosaccharides rapidly to mixtures of smaller xylo-oligosaccharides, with xylobiose and xylose being the preponderant final products. The anomeric configuration of the products was inverted, in contrast to the behavior of most other carbohydrases that initially produce mixtures of oligosaccharides. This enzyme is a glycoprotein having an amino acid composition high in acidic residues. Its molecular weight is 20,800 and its isoelectric point is at pH 6.7. Optimal pH values for activity and stability are between 4 and 6 and, in a 20-min assay, maximal activity is attained at 55°.

Crystal structure and evolution of a prokaryotic glucoamylase.

The first crystal structures of a two-domain, prokaryotic glucoamylase were determined to high resolution from the clostridial species Thermoanaerobacterium thermosaccharolyticum with and without acarbose. The N-terminal domain has 18 antiparallel strands arranged in beta-sheets of a super-beta-sandwich. The C-terminal domain is an (alpha/alpha)(6) barrel, lacking the peripheral subdomain of eukaryotic glucoamylases. Interdomain contacts are common to all prokaryotic Family GH15 proteins. Domains similar to those of prokaryotic glucoamylases in maltose phosphorylases (Family GH65) and glycoaminoglycan lyases (Family PL8) suggest evolution from a common ancestor. Eukaryotic glucoamylases may have evolved from prokaryotic glucoamylases by the substitution of the N-terminal domain with the peripheral subdomain and by the addition of a starch-binding domain.

High-performance anion-exchange chromatography of sugars and sugar alcohols on quaternary ammonium resins under alkaline conditions

Abstract Ninety-three sugars and sugar alcohols of one to four monosaccharide units were subjected to high-performance anion-exchange chromatography on three quaternary ammonium columns of the same size and virtually the same composition under identical eluent flow rates (1 mL·min−1) and compositions (aqueous 0.1 n NaOH). Although the columns had very different histories of use, their different capacity factors for individual carbohydrates can be linearly correlated. Those for monosaccharides are lowest for sugar alcohols, and are higher and roughly similar for analogous aldoses and ketoses. There is a general trend, strongest with sugar alcohols, for capacity factors to increase withincreasing numbers of carbon atoms. Increasing numbers of hydroxyl groups on residues of similar structure lead to increasing capacity factors. For members of homologous oligosaccharides series, capacity factors increase in a regular and predictable manner with chain length. Peak areas for different sugars generated by a differential refractometer are roughly correlated with mass concentrations; with a pulsed amperometric detector, there are equally rough correlations with either molar or mass concentration.