The aminopeptidase from Streptomyces griseus (SGAP) has been cloned and expressed in Escherichia coli. By growing the cells in the presence of 1 M sorbitol at 18°C, the protein was obtained in a soluble and active form. The amino acid sequence of the recombinant SGAP contained four amino acids differing from the previously published sequence. Resequencing of the native protein indicated that asparagines 70 and 184 are in fact aspartic acids as in the recombinant protein.Based on the crystal structure of SGAP, Glu131 and Tyr246 were proposed to be the catalytic residues. Replacements of Glu131 resulted in loss of activity of 4-5 orders of magnitude, consistent with Glu131 acting as the general base residue. Mutations in Tyr246 resulted in about 100-fold reduction of activity, suggesting that this residue is involved in the stabilization of the transition state intermediate.

Aminopeptidases are exopeptidases that catalyze the removal of N-terminal amino acids from peptides; they are found in bacteria, plants and mammalian tissues. Many aminopeptidases are metallo-enzymes, containing two catalytic transition metals (usually zinc) in their active site [1][2][3]. The activity of these enzymes is associated with many central biological processes, such as protein maturation, protein degradation, hormone level regulation, angiogenesis and cell-cycle control [4][5][6][7][8]. Not surprisingly, aminopeptidases play an important role in many pathological conditions, including cancer, cataract, cystic fibrosis and HIV infection. Indeed, antitumor drugs such as ovalicin and fumagillin were found to inhibit aminopeptidases. In this regard, the natural inhibitor for aminopeptidases, bestatin, was recently shown to significantly decrease HIV infection by inhibiting aminopeptidase activity [9][10][11]. Aminopeptidases can be classified into clans and families based on their amino acid sequence homology. Clan M contains mainly metallopeptidase families, one of which is M28. 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 kJAEmol) 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°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 kJAEmol )1 in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis.Abbreviations AAP, Aeromonas proteolytica aminopeptidase; blLAP, bovine lens leucine aminopeptidase; Leu-pNA, leucine-para-nitroanilide; SGAP, Streptomyces griseus aminopeptidase.

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus 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 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high thermal and pH stabilities, which are required for a number of biotechnological and industrial applications.

Keywords: Amine ligands / Chelates / Ligand design / Stability constantsThe protonation and metal complex formation of the branched pentaamine ligand 2,2,6,6-tetrakis(aminomethyl)-

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.