True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.

Penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) is widely distributed among microorganisms, including bacteria, yeast and filamentous fungi. It is used on an industrial scale for the production of 6-aminopenicillanic acid, the starting material for the synthesis of semi-synthetic penicillins. Its in vivo role remains unclear, however, and the observation that expression of the Escherichia coli enzyme in vivo is regulated by both temperature and phenylacetic acid has prompted speculation that the enzyme could be involved in the assimilation of aromatic compounds as carbon sources in the organism's free-living mode. The mature E. coli enzyme is a periplasmic 80K heterodimer of A and B chains (209 and 566 amino acids, respectively) synthesized as a single cytoplasmic precursor containing a 26-amino-acid signal sequence to direct export to the cytoplasm and a 54-amino-acid spacer between the A and B chains which may influence the final folding of the chains. The N-terminal serine of the B chain reacts with phenylmethylsulphonyl fluoride, which is consistent with a catalytic role for the serine hydroxyl group. Modifying this serine to a cysteine inactivates the enzyme, whereas threonine, arginine or glycine substitution prevents in vivo processing of the enzyme, indicating that this must be an important recognition site for cleavage. Here we report the crystal structure of penicillin acylase at 1.9 A resolution. Our analysis shows that the environment of the catalytically active N-terminal serine of the B chain contains no adjacent histidine equivalent to that found in the serine proteases. The nearest base to the hydroxyl of this serine is its own alpha-amino group, which may act by a new mechanism to endow the enzyme with its catalytic properties.

Cellulose, a polymer of beta-1,4-linked glucose residues, is the major polysaccharide component of plant cell walls and the most abundant biopolymer. The underlying mechanisms of the enzymatic degradation of cellulose are of increasing commercial and ecological significance. Endoglucanase V, from the cellulolytic soil hyphomycete Humicola insolens, is an endocellulase, the catalytic core of which consists of 210 amino acids and is known to hydrolyze the beta-1,4 links with inversion of configuration at the anomeric carbon. The major products of cellulose hydrolysis are cellobiose and cellotriose. The crystal structures of the endoglucanase V (EGV) from H. insolens, in native, product (cellobiose), inactive mutant (D10N), and oligosaccharide-bound [(D10N)-cellohexaose] forms, have been determined at resolutions of 1.9 A or better. EGV consists of a six-stranded beta-barrel domain with long interconnecting loops. A 40 A groove exists along the surface of the enzyme, and this contains the catalytic residues, Asp 10 and Asp 121. The two catalytic aspartates sit to either side of the substrate binding groove in an ideal conformation for facilitating cleavage by inversion, their carboxyl groups being separated by approximately 8.5 A. The complex between substrate and inactive mutant reveals excellent density for an oligosaccharide in six of the enzyme's seven substrate binding subsites. No sugar moiety, however, is seen bound to the -1 subsite at the point of cleavage. The geometry of the cleavage site suggests that the enzyme would favor the binding of sugars with an elongated glycosidic bond, as found in the transition state, as opposed to the binding of substrate. The oligosaccharide complexes reveal solvent water suitably placed for participation in a single displacement reaction as first suggested by Koshland in 1953 [Koshland, D. E. (1953) Biol. Rev. 28, 416-436]. A large conformational change takes place upon substrate binding. This "lid flipping" has the effect of increasing the hydrophobic environment of the catalytic proton donor, enclosing the active site at the point of cleavage, and bringing a third aspartate (Asp 114) in close proximity to the substrate. Site-directed mutagenesis of the catalytic residues has been used to confirm their significance in catalysis.

Cellulose is the major polysaccharide component of plant cell walls and is the most abundant organic compound on the planet. A number of bacterial and fungal organisms can use cellulose as a food source, possessing cellulases (cellobiohydrolases and endoglucanases) that can catalyse the hydrolysis of the beta-(1,4) glycosidic bonds. They can be classified into seven distinct families. The three-dimensional structures of members of two of these families are known. Here we report the structure of a third cellulase, endoglucanase V, whose sequence is not represented in any of the above families. The enzyme is structurally distinct from the previously determined cellulases but is similar to a recently characterized plant defence protein. The active site region resembles that of lysozyme, despite the lack of structural similarity between these two enzymes.

The structure of the catalytic core of the endoglucanase V (EGV) from Humicola insolens has been determined by the method of multiple isomorphous replacement at 1.5 A, resolution. The final model, refined with X-PLOR and PROLSQ, has a crystallographic R factor of 0.163 (Rfree = 0.240) with deviations from stereochemical target values of 0.012 ,~ and 0.037 ° for bonds and angles, respectively. The model was further refined with SHELXL, including anisotropic modelling of the protein-atom temperature factors, to give a final model with an R factor of 0.105 and an Rfre~ of 0.154. The initial isomorphous replacement electron-density map was poor and uninterpretable but was improved by the use of synchrotron data collected at a wavelength chosen so as to optimize the f" contribution of the anomalous scattering from the heavy atoms. The structure of H. insolens EGV consists of a six-stranded/3-barrel domain, similar to that found in a family of plant defence proteins, linked by a number of disulfide-bonded loop regions. A long open groove runs across the surface of the enzyme either side of which lie the catalytic aspartate residues. The 9/~ separation of the catalytic carboxylate groups is consistent with the observation that EGV catalyzes the hydrolysis of the cellulose ,0(144) links with inversion of configuration at the anomeric CI atom. This structure is the first representative from the glycosyl hydrolase family 45.