Rotem Gilboa

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.

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.

Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 A resolution.

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.

Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6.

The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43,808 and pI of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme. The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive. The crystals belong to a primitive trigonal crystal system (space group P3(1) or P3(2)) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A. These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution. The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays. A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions.

Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus

The catalytic mechanism underlying the aminopeptidase from Streptomyces griseus (SGAP) was investigated. pH‐dependent activity profiles revealed the enthalpy of ionization for the hydrolysis of leucine‐para‐nitroanilide by SGAP. The value obtained (30 ± 5 kJ·mol−1) is typical of a zinc‐bound water molecule, suggesting that the zinc‐bound water/hydroxide molecule acts as the reaction nucleophile. Fluoride was found to act as a pure noncompetitive inhibitor of SGAP at pH values of 5.9–8 with a Ki of 11.4 mm at pH 8.0, indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme–substrate complex. pH‐dependent pKi experiments resulted in a pKa value of 7.0, suggesting a single deprotonation step of the catalytic water molecule to an hydroxide ion. The number of proton transfers during the catalytic pathway was determined by monitoring the solvent isotope effect on SGAP and its general acid–base mutant SGAP(E131D) at different pHs. The results indicate that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. The role of Glu131 in binding and catalysis was assessed by determining the catalytic constants (Km, kcat) over a temperature range of 293–329 °K for both SGAP and the E131D mutant. For the binding step, the measured and calculated thermodynamic parameters for the reaction (free energy, enthalpy and entropy) for both SGAP and the E131D mutant were similar. By contrast, the E131D point mutation resulted in a four orders of magnitude decrease in kcat, corresponding to an increase of 9 kJ·mol−1 in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis.

Binding of inhibitory aromatic amino acids to Streptomyces griseus aminopeptidase.

The bacterial aminopeptidase isolated from the extracellular extract of Streptomyces griseus (SGAP) is a double-zinc exopeptidase with a high preference for large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), is heat-stable, displays a high and efficient catalytic turnover and its activity is modulated by calcium ions. Several free amino acids were found to inhibit the activity of SGAP in the millimolar concentration range and can therefore serve for the study of binding of both inhibitors and reaction products. The current study is focused on the X-ray crystallographic analysis of the SGAP complexes with L-tryptophan and p-iodo-L-phenylalanine, both at 1.30 A resolution. These two bulky inhibitory amino acids were found to bind to the active site of SGAP in very similar positions and orientations. Both of them bind to the two active-site zinc ions via their free carboxylate group, while displacing the zinc-bound water/hydroxide that is present in the native enzyme. Further stabilization of the binding of the amino-acid carboxylate group is achieved by its relatively strong interactions with the hydroxyl group of Tyr246 and the carboxylate group of Glu131. The binding is also stabilized by three specific hydrogen bonds between the amine group of the bound amino acid and enzyme residues Glu131, Asp160 and Arg202. These consistent interactions confirm the key role of these residues in the specific binding of the free amine of substrates and products, as proposed previously. The phenyl ring of Phe219 of the enzyme is involved in stacking interactions with the corresponding aromatic ring of the bound affector. This interaction seems to be important for the binding and orientation of the aromatic side chain within the specificity pocket. These structural results correlate well with the results obtained for the complexes of SGAP with other inhibitory amino acids and support the general catalytic mechanism proposed for this and related enzymes.

Crystallization and preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from Sacchromyces cerevisiae (baker's yeast)

Two related and not thoroughly resolved issues in biochemistry concern the role, if any, of enzyme surfaces in routine metabolism and the method by which metabolic intermediates move between enzyme active sites during multi-step degradation or synthesis. An important enzyme for which a detailed three-dimensional structural analysis has been initiated is yeast glyceraldehyde 3-phosphate dehydrogenase (yGAP-DH). This enzyme is active as a tetramer of total molecular weight of 145 kDa and requires nicotinamide adenine dinucleotide (NAD+) as cofactor. In this report, the crystallization and preliminary crystallographic characterization of several crystal forms of yGAP-DH are described. Of the five distinct crystal forms, the most suitable was found to contain the holo-enzyme, and the crystals were grown by the vapor-diffusion method using polyethylene glycol 6000 as precipitant, sodium acetate as buffer (pH 4.6), and NAD+ and dithiothreitol as additives. The crystals belong to the orthorhombic space group P21212, with cell dimensions of a = 87.33, b = 96.11 and c = 115.34 A. These crystals are mechanically strong, relatively stable in the X-ray beam and diffract X-rays (from a normal rotating-anode radiation source) to better than 2 A resolution. A full 2.1 A resolution diffraction data set (98% completion) has been measured. The three-dimensional structures of related GAP-DH enzymes from several other sources have been determined and reported, and are available for a molecular replacement structure solution.

Preliminary crystallographic characterization of BSAP, an extracellular aminopeptidase from Bacillus subtilis

The extracellular aminopeptidase from Bacillus subtilis (BSAP) has recently been cloned, overexpressed and purified from Escherichia coli. It is a monomer with a molecular weight of 46 425 Da, consisting of 425 amino-acid residues and a double-zinc catalytic centre. The recombinant enzyme was found to be stable for 20 min at 353 K, to function optimally in the pH range 8-9 and to prefer basic and large hydrophobic N-terminal amino acids in peptide and protein substrates. As such, this enzyme can be used as a representative model for structural, functional and mechanistic studies of monomeric double-zinc aminopeptidases, many of which have been found to be involved in medically important biological activities. In this report, the crystallization and preliminary crystallographic characterization of wild-type BSAP are described. Two different crystal forms are reported, of which the hexagonal form H2 is the more suitable for structural study, with average unit-cell dimensions a = b = 226.5, c = 42.8 A. A full diffraction data set has been collected from such a crystal of the native enzyme (2.2 A resolution, 91.2% completeness, R(merge) = 7.1%). A multiwavelength anomalous diffraction (MAD) data set was collected on native (zinc-containing) BSAP at three wavelengths around the zinc absorption edge (peak data set at 2.5 A resolution, 98.8% completeness, R(merge) = 5.3%). These diffraction data were collected at 95-100 K using a synchrotron X-ray source and a CCD area detector. The data are currently being used to obtain crystallographic phasing and to determine the detailed three-dimensional structure of the enzyme.