Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.

The 2.0 A crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals a fold similar to that of tyrosine hydroxylase. It provides the first structural view of where mutations occur and a rationale to explain molecular mechanisms of the enzymatic phenotypes in the autosomal recessive disorder phenylketoneuria.

In this paper, we describe the structure of chitinase B from Serratia marcescens, which consists of a catalytic domain with a TIM-barrel fold and a 49-residue C-terminal chitin-binding domain. This chitinase is the first structure of a bacterial exochitinase, and it represents one of only a few examples of a glycosyl hydrolase structure having interacting catalytic and substrate-binding domains. The chitin-binding domain has exposed aromatic residues that contribute to a 55-Å long continuous aromatic stretch extending into the active site. Binding of chitin oligomers is blocked beyond the ؊3 subsite, which explains why the enzyme has chitotriosidase activity and degrades the chitin chain from the nonreducing end. Comparison of the chitinase B structure with that of chitinase A explains why these enzymes act synergistically in the degradation of chitin.T he degradation of abundant insoluble carbohydrate polymers such as cellulose and chitin is achieved in nature with the help of batteries of glycosyl hydrolases with different substrate preferences and product specificities. For example, the degradation of chitin, a linear polysaccharide of ␤(1, 4)-linked N-acetylglucosamine (GlcNAc) residues, by the soil bacterium S. marcescens involves at least four enzymes (the exo-and endochitinases ChiA, ChiB, and ChiC, and an N-acetylglucosaminidase) (1-4). In addition to a catalytic domain, most enzymes involved in cellulose and chitin degradation usually contain one or more domains that are involved in substrate binding (refs. 5-7; see also http:͞͞afmb.cnrsmrs.fr͞pedro͞DB͞ncmCBM12.html). Removal of such domains often results in enzymes that are still active but display severely impaired binding to polymeric substrates (see examples in refs. 7 and 8). For cellulases, there is abundant structural information for a variety of catalytic domains and for isolated carbohydrate-binding domains (6, 9), but there is only one available crystal structure of a catalytic domain together with a (143-residue) CeBD (10).Chitinases belong to families 18 and 19 of the glycosyl hydrolases (9). The catalytic domain of family 18 chitinases has a TIM-barrel fold (2, 11) and includes a conserved glutamate residue that presumably acts as an acid during catalysis (Glu144 in ChiB; Fig. 1 ; refs. 12-14). Catalysis proceeds with retention of the anomeric configuration, which is achieved by a mechanism in which the carbonyl oxygen of the N-acetyl group of the Ϫ1 sugar (nomenclature according to ref. 15) acts as nucleophile (12)(13)(14). Judged from their sequences, most family 18 chitinases, including ChiA, ChiB, and ChiC from S. marcescens, contain domains putatively involved in the interaction with chitin (5,7,8,16). Perrakis et al. (2,5) have determined the structure of complete ChiA, revealing the location of a 114-residue domain with a fibronectin III-like fold that most likely participates in chitin-binding (2,5,8). On the basis of sequence analyses, ChiB has been suggested to consist of a catalytic domain followed by a putative linker region an...

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The structure of PLD provides insight into the structure and mode of action of not only bacterial, plant and mammalian PLDs, but also of a variety of enzymes as diverse as cardiolipin synthases, phosphatidylserine synthases, toxins, endonucleases, as well as poxvirus envelope proteins having a so far unknown function. The common features of these enzymes are that they can bind to a phosphodiester moiety, and that most of these enzymes are active as bi-lobed monomers or dimers.

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The aromatic amino acid hydroxylases represent a superfamily of structurally and functionally closely related enzymes, one of those functions being reversible inhibition by catechol derivatives. Here we present the crystal structure of the dimeric catalytic domain (residues 117-424) of human phenylalanine hydroxylase (hPheOH), cocrystallized with various potent and well-known catechol inhibitors and refined at a resolution of 2.0 A. The catechols bind by bidentate coordination to each iron in both subunits of the dimer through the catechol hydroxyl groups, forming a blue-green colored ligand-to-metal charge-transfer complex. In addition, Glu330 and Tyr325 are identified as determinant residues in the recognition of the inhibitors. In particular, the interaction with Glu330 conforms to the structural explanation for the pH dependence of catecholamine binding to PheOH, with a pKa value of 5.1 (20 degreesC). The overall structure of the catechol-bound enzyme is very similar to that of the uncomplexed enzyme (rms difference of 0.2 A for the Calpha atoms). Most striking is the replacement of two iron-bound water molecules with catechol hydroxyl groups. This change is consistent with a change in the ligand field symmetry of the high-spin (S = 5/2) Fe(III) from a rhombic to a nearly axial ligand field symmetry as seen upon noradrenaline binding using EPR spectroscopy [Martinez, A., Andersson, K. K., Haavik, J., and Flatmark, T. (1991) Eur. J. Biochem. 198, 675-682]. Crystallographic comparison with the structurally related rat tyrosine hydroxylase binary complex with the oxidized cofactor 7,8-dihydrobiopterin revealed overlapping binding sites for the catechols and the cofactor, compatible with a competitive type of inhibition of the catechols versus BH4. The comparison demonstrates some structural differences at the active site as the potential basis for the different substrate specificity of the two enzymes.