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 K(i) 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 pK(i) experiments resulted in a pK(a) 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 (K(m), k(cat)) over a temperature range of 293-329 degrees 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 k(cat), 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.

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.

Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis.

Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.

Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA.

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.

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 beta-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 A resolution. The crosslink is derived from a Schiff base intermediate that precedes beta-elimination and is stabilized by reduction with NaBH(4). 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.