Von Willebrand factor (VWF)2 is a large multimeric plasma glycoprotein that mediates tethering and adhesion of circulating platelets at sites of vascular injury (1). Following endothelial damage, plasma VWF binds to the exposed sub-endothelial collagen (2). Once immobilized, the shear forces of the flowing blood induce a conformational transition that unravels the VWF molecule (3). This in turn exposes the binding sites for glycoprotein (Gp)Ib␣, part of the GpIb-IX-V receptor on the surface of circulating platelets that confer platelet-tethering function (4). Once tethered, platelets become activated and subsequently present GpIIb/IIIa on their surface, which makes a tighter, more stable interaction with both VWF and fibrinogen. Activated platelets provide the phosphatidylserine-rich surface critical for the assembly of the procoagulant enzyme complexes that lead to the generation of thrombin (5).VWF is expressed by endothelial cells and megakaryocytes (6). It is synthesized as 250-kDa monomers, which undergo intracellular processing-glycosylation, multimerization, and propeptide removal-that leads to formation of mature VWF multimers (7). Although much of endothelial VWF is constitutively secreted into the blood as multimers of varying size, a proportion is stored either within Weibel-Palade bodies in endothelial cells or within ␣-granules in platelets (8). These stored pools, which can be released upon specific stimulation, contain predominantly hyper-reactive "ultra-large" VWF that can exceed 2 ϫ 10 4 kDa. A wide range of VWF multimers (500 -20,000 kDa) are found in normal plasma that differ only by the number of constituent VWF units. The largest VWF multimers unravel more readily in response to shear forces and contain more platelet binding sites. These species therefore confer the greatest hemostatic potential. The size of plasma VWF, and thus its platelet-tethering function, is regulated to prevent aberrant/ spontaneous platelet-rich thrombus formation. VWF multimeric size is modulated by the plasma metalloproteinase ADAMTS13, which cleaves at a single site in the VWF A2 domain between Tyr 1605 and Met 1606 (9). This proteolysis can only proceed once VWF has been unraveled, either by rheological forces or in vitro in the presence of denaturants, both of which induce the exposure of the A2 domain scissile bond (10). Physiologically, this occurs upon VWF secretion from endothelial cells, limiting the thrombogenic potential of newly secreted ultra-large VWF by converting it into smaller multimeric forms (11). The nature of ADAMTS13 recognition of the A2 domain is poorly understood. In part, this is because of complex folding and interdependence of the A1-A2-A3 domains. This has been illustrated by proteolysis studies conducted with isolated domain fragments and certain VWF mutations that confer type 2A (group II) von Willebrand disease (VWD) (12)(13)(14). The latter are proximate or within the A2 domain and manifest enhanced ADAMTS13-dependent VWF proteolysis. This is thought to be because of structural chan...

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase α-subunit (XamoA); aamB, encoding the 88-residue oxygenase γ-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase β-subunit (XamoE); andaamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of ς54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase α-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase fromRhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacterstrain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture ofo-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studiesXanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of theaam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.

The epoxide hydrolase (EH) from Corynebacterium sp. C12, which grows on cyclohexene oxide as sole carbon source, has been purified to homogeneity in two steps, involving anion exchange followed by hydrophobic-interaction chromatography. The purified enzyme is multimeric (probably tetrameric) with a subunit size of 32 140 Da. The gene encoding Corynebacterium EH was located on a 3.5-kb BamHI fragment of C12 chromosomal DNA using a DNA probe generated by PCR using degenerate primers based on the N-terminal and an internal amino acid sequence. Sequencing and database comparison of the predicted amino acid sequence of Corynebacterium EH shows that it is similar to mammalian and plant soluble EH, and the recently published sequence of epichlorohydrin EH from Agrobacterium radiobacter AD1 [Rink, R., Fennema, M., Smids, M., Dehmel, U. & Janssen, D. B. (1997) J. Biol. Chem. 272, 14 650Ϫ14 657), particularly around the catalytic site. All of these proteins belong to the A/β-hydrolase-fold family of enzymes. Similarity to the mammalian microsomal EH is weaker.Keywords : epoxide hydrolase; Corynebacterium C12 ; cyclohexene oxide ; A/β hydrolase fold.Extensive studies on the mammalian soluble (cytosolic) and chemical studies of soluble EH have demonstrated the formation microsomal epoxide hydrolases (EH), enzymes with a central of intermediate aspartate adducts , and role in detoxification (Oesch, 1973;Hammock et al., 1997), single-turnover experiments with microsomal EH (Lacourciere have established that they belong to a family of proteins that and Armstrong, 1993) have confirmed that the hydroxyl group share regions of amino acid sequence similarity with the active transferred to the product derives from the enzyme and not disite of bacterial haloalkane dehalogenase . rectly from water. These experiments also demonstrated the laThe three-dimensional structure of the latter was characterised belling of a specific aspartate residue during turnover, by H 2 18 O. by Verschueren et al. and coworkers (1993) who demonstrated Site-directed-mutagenesis studies with soluble EH (Pinot et al., that the enzyme belongs to the A/β-hydrolase-fold class of en-1995; Arand et al., 1996) have also confirmed the essential role zymes and employs a two-step mechanism involving initial for-of a histidine and aspartate pair, predicted by sequence alignmation of an aspartate ester, which is hydrolysed subsequently ment with haloalkane dehalogenase and other A/β hydrolase fold by a water molecule activated by a histidine-aspartate pair. enzymes, to be involved in promoting the hydrolytic step. While the core structure of the A/β hydrolase family, formed by Genes encoding the soluble EH have been cloned from a sheet of parallel β strands sandwiched by A helices (Ollis et mammals and plants (Beetham et al., 1995), and microsomal EH al., 1992), supports the catalytic triads of hydrolytic enzymes genes have been cloned from mammals and an insect source such as lipase and serine carboxypeptidase, a distinguishing fea- (Wojtasek and Prestwich, 1996). W...

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear nonhaem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase ( s 60% sequence similarity) and methane monooxygenase ( s 40% sequence similarity) and that the necessary sequence motifs associated with iron coordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly K Khelical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the coordinates of the methane monooxygenase K K-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.z 1998 Federation of European Biochemical Societies.

Nitrile hydratase from Brevibacterium sp. R312 was purified to homogeneity. The isoelectric point was 5.75. The two kinds of subunits were separated by reverse phase HPLC and their N-terminal amino acid sequences were found to be identical to those of Rhodococcus sp. N-774 nitrile hydratase.