Gil Shoham

Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate

Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine‐DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential β‐elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 Å resolution. The crosslink is derived from a Schiff base intermediate that precedes β‐elimination and is stabilized by reduction with NaBH4. Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site‐directed mutagenesis studies, we propose a catalytic mechanism for Nei.

Crystal structure and snapshots along the reaction pathway of a family 51 α‐l‐arabinofuranosidase

High‐resolution crystal structures of α‐L‐arabinofuranosidase from Geobacillus stearothermophilus T‐6, a family 51 glycosidase, are described. The enzyme is a hexamer, and each monomer is organized into two domains: a (β/α)8‐barrel and a 12‐stranded β sandwich with jelly‐roll topology. The structures of the Michaelis complexes with natural and synthetic substrates, and of the transient covalent arabinofuranosyl—enzyme intermediate represent two stable states in the double displacement mechanism, and allow thorough examination of the catalytic mechanism. The arabinofuranose sugar is tightly bound and distorted by an extensive network of hydrogen bonds. The two catalytic residues are 4.7 Å apart, and together with other conserved residues contribute to the stabilization of the oxocarbenium ion‐like transition state via charge delocalization and specific protein—substrate interactions. The enzyme is an anti‐protonator, and a 1.7 Å electrophilic migration of the anomeric carbon takes place during the hydrolysis.

Structural characterization of the Fpg family of DNA glycosylases

Until recently, the Fpg family was the only major group of DNA glycosylases for which no structural data existed. Prototypical members of this family, found in eukaryotes as well as prokaryotes, have now been crystallized as free proteins and as complexes with DNA. In this review, we analyze the available structural information for formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei). Special emphasis is placed on mechanisms by which these enzymes recognize and selectively excise cognate lesions from oxidatively damaged DNA. The problem of lesion recognition is considered in two parts: how the enzyme efficiently locates a single lesion embedded in a vast excess of DNA; and how the lesion is accommodated in a pocket near the active site of the enzyme. Although all crystal structures reported to date for the Fpg family lack the damaged base, functionally important residues that participate in DNA binding and enzyme catalysis have been clearly identified and other residues, responsible for substrate specificity, have been inferred.

The identification of the acid–base catalyst of α-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase

The α‐L‐arabinofuranosidase from Geobacillus stearothermophilus T‐6 (AbfA T‐6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid–base catalytic residue. AbfA T‐6 exhibits residual activity towards aryl β‐D‐xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid–base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid–base catalyst of AbfA T‐6.

Structure–specificity relationships of an intracellular xylanase from Geobacillus stearothermophilus

Geobacillus stearothermophilus T-6 is a thermophilic Gram-positive bacterium that produces two selective family 10 xylanases which both take part in the complete degradation and utilization of the xylan polymer. The two xylanases exhibit significantly different substrate specificities. While the extracellular xylanase (XT6; MW 43.8 kDa) hydrolyzes the long and branched native xylan polymer, the intracellular xylanase (IXT6; MW 38.6 kDa) preferentially hydrolyzes only short xylo-oligosaccharides. In this study, the detailed three-dimensional structure of IXT6 is reported, as determined by X-ray crystallography. It was initially solved by molecular replacement and then refined at 1.45 A resolution to a final R factor of 15.0% and an R(free) of 19.0%. As expected, the structure forms the classical (alpha/beta)(8) fold, in which the two catalytic residues (Glu134 and Glu241) are located on the inner surface of the central cavity. The structure of IXT6 was compared with the highly homologous extracellular xylanase XT6, revealing a number of structural differences between the active sites of the two enzymes. In particular, structural differences derived from the unique subdomain in the carboxy-terminal region of XT6, which is completely absent in IXT6. These structural modifications may account for the significant differences in the substrate specificities of these otherwise very similar enzymes.

A correlation between the proton stretching vibration red shift and the hydrogen bond length in polycrystalline amino acids and peptides.

The FTIR spectra of pure and isotopically diluted (H/D and D/H) polycrystalline L-glutamine, L-hystidine, L-tyrosine, DL-serine, L-threonine, di-, tri-glycine and di-glycine x HCl x H2O salt were measured in the range 4000-2000 cm(-1) at temperatures from 300 to 10 K. The frequencies of decoupled proton stretching mode bands upsilon1, which can be observed only at low temperature, were used for estimation of the of upsilon1-bands red shift, which occurs upon formation of H-bonds involving ionized NH3+ and/or peptide HN-CO groups. The empirical correlation between the red shift and H-bond length, which was found previously for binary gas phase H-bonded complexes, carbohydrates and nucleosides [M. Rozenberg, A. Loewenschuss and Y. Marcus, Phys. Chem. Chem. Phys., 2000, 2, 2699-2702; M. Rozenberg, C. Jung and G. Shoham, Phys. Chem. Chem. Phys., 2003, 5, 1533-1535], was now extended to H-bonded networks in polycrystalline amino acids and peptides. The energies of the different H-bonds present in the crystalline structures could also be successfully estimated from the well-established empirical correlation [A. V. Iogansen, Spectrochim. Acta, 1999, A55, 1585-1612] between this property and the red shifts of the corresponding upsilon1 mode bands.

Crystal Structures of Geobacillus stearothermophilus {alpha}-Glucuronidase Complexed with Its Substrate and Products: MECHANISTIC IMPLICATIONS.

α-Glucuronidases cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the α-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the α-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-d-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp364 and Glu392, activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu285 cannot be resolved in some of the structures but could be visualized in its “open” and “closed” (catalytic) conformations in other structures. The protonated state of Glu285 is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism

Streptomyces griseus aminopeptidase (SGAP) is a double‐zinc exopeptidase with a high preference toward large hydrophobic amino‐terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X‐ray crystallographic analysis of the SGAP complexes with methionine (1.53 Å resolution), leucine (1.70 Å resolution), and phenylalanine (1.80 Å resolution). These three high‐resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme‐substrate and enzyme‐product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme‐substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc‐bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP. Proteins 2001;44:490–504.

A Universal Screening Assay for Glycosynthases: Directed Evolution of Glycosynthase XynB2(E335G) Suggests a General Path to Enhance Activity

Glycosynthases are catalytic mutants of mainly retaining glycoside hydrolases that catalyze the synthesis of oligosaccharides from their corresponding glycosyl-fluoride donors and suitable acceptors. Here we describe the development of a general, high-throughput screening procedure for glycosynthase activity, which is based on the release of hydrofluoric acid, a by-product of all glycosynthase reactions. This assay is sensitive, does not require the synthesis of special chromophoric or modified substrates, and, most importantly, is applicable for all glycosynthases. We used this screening procedure on error-prone PCR libraries to isolate improved glycosynthase variants of XynB2(E335G) glycosynthase, a family 52 beta-xylosidase from Geobacillus stearothermophilus. The improved variants exhibited higher K(M) values toward the acceptor and the donor, suggesting that enzyme-product release is rate determining for k(cat).

Glycosynthase activity of Geobacillus stearothermophilus GH52 β-xylosidase : Efficient synthesis of xylooligosaccharides from α-D-xylopyranosyl fluoride through a conjugated reaction

Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile‐deficient mutant of a glycoside hydrolase family 52 β‐xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using α‐D‐xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self‐condensation reaction of α‐D‐xylopyranosyl fluoride, providing mainly α‐D‐xylobiosyl fluoride. The self‐condensation kinetics exhibited apparent classical Michaelis–Menten behavior, with kinetic constants of 1.3 s−1 and 2.2 mM for kcat and KM(acceptor), respectively, and a kcat/KM(acceptor) value of 0.59 s−1 mM−1. When the β‐xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high‐molecular‐weight xylooligomers were readily obtained from the affordable α‐D‐xylopyranosyl fluoride as the sole substrate.