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Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.

This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8 A resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8 A resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

The crystal structure of carboxypeptidase T from Thermoactirzomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the threedimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed p sheet. The active site is located at the C-edge of the central (parallel) part of the B sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.Metallocarboxypeptidases are exopeptidases which contain a zinc ion in the active centre and catalyze the hydrolysis of C-terminal amino acids from polypeptide substrates. Extensive studies of bovine pancreatic carboxypeptidase A (CPA) by different techniques including crystallography, spectroscopy, kinetics and site-directed mutagenesis resulted in the understanding of the general principles of the catalytic mechanism (Christianson and Lipscomb, 1989). Three-dimensional structures have been determined for pancreatic carboxypeptidases A (Rees et al., 1983;Kobe and Goldsmith, 1990) and B (Schmid and Herriott, 1976; Coll et al., 1991) which share a common polypeptide fold but differ in substrate specificity (Hartsuck and Lipscomb, 1971 ;Folk, 1971 polypeptide chain of 326 amino acids and contains one zinc ion (Osterman et al., 1984). The amino acid sequence of CPT, deduced from the gene encoding the protein (Smulevitch et al., 1991), shows only moderate similarity with the CPA and CPB sequences; 30% and 27% identical residues, respectively. CPT exhibits an unusual 'dual'-substrate specificity combining features of both CPA and CPB; it is able to split off hydrophobic and basic amino acids with comparable efficiency (Osterman et al., 1984). As other proteins from Thermoactinomyces, CPT has an increased thermal stability in the presence of calcium ions (Osterman et al., 1984).Structure/functional studies of CPT provide a possibility to study the general principles of catalysis by Zn-carboxypeptidases. On the basis of the comparative primary-structure analysis, all Zn-carboxypeptidases were divided into two groups ; ,,digestive,, and ,,regulatory,, enzymes (Osterman et al., 1992). Pancreatic CPA and CPB appear to be structurally ...

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