In higher organisms the formation of the steroid scaffold is catalysed exclusively by the membrane-bound oxidosqualene cyclase (OSC; lanosterol synthase). In a highly selective cyclization reaction OSC forms lanosterol with seven chiral centres starting from the linear substrate 2,3-oxidosqualene. Valuable data on the mechanism of the complex cyclization cascade have been collected during the past 50 years using suicide inhibitors, mutagenesis studies and homology modelling. Nevertheless it is still not fully understood how the enzyme catalyses the reaction. Because of the decisive role of OSC in cholesterol biosynthesis it represents a target for the discovery of novel anticholesteraemic drugs that could complement the widely used statins. Here we present two crystal structures of the human membrane protein OSC: the target protein with an inhibitor that showed cholesterol lowering in vivo opens the way for the structure-based design of new OSC inhibitors. The complex with the reaction product lanosterol gives a clear picture of the way in which the enzyme achieves product specificity in this highly exothermic cyclization reaction.

Halogen bonding triggers activity: Increasing binding affinity was observed for a series of covalent human Cathepsin L inhibitors by exchanging an aryl ring H atom with Cl, Br, and I, which undergo halogen bonding with the CO group of Gly61 in the S3 pocket of the enzyme. Fluorine, in contrast, strongly avoids halogen bonding (see scheme). The strong distance and angle dependence of halogen bonding was confirmed for biological systems.

The atomic structures of two proteins in the histidine biosynthesis pathway consist of beta/alpha barrels with a twofold repeat pattern. It is likely that these proteins evolved by twofold gene duplication and gene fusion from a common half-barrel ancestor. These ancestral domains are not visible as independent domains in the extant proteins but can be inferred from a combination of sequence and structural analysis. The detection of subdomain structures may be useful in efforts to search genome sequences for functionally and structurally related proteins.

Inhibition of dipeptidyl peptidase IV (DPP-IV), the main glucagon-like peptide 1 (GLP1)-degrading enzyme, has been proposed for the treatment of type II diabetes. We expressed and purified the ectodomain of human DPP-IV in Pichia pastoris and determined the X-ray structure at 2.1 A resolution. The enzyme consists of two domains, the catalytic domain, with an alpha/beta hydrolase fold, and a beta propeller domain with an 8-fold repeat of a four-strand beta sheet motif. The beta propeller domain contributes two important functions to the molecule that have not been reported for such structures, an extra beta sheet motif that forms part of the dimerization interface and an additional short helix with a double Glu sequence motif. The Glu motif provides recognition and a binding site for the N terminus of the substrates, as revealed by the complex structure with diprotin A, a substrate with low turnover that is trapped in the tetrahedral intermediate of the reaction in the crystal.

The enzymes N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (HisA) and phosphoribosylanthranilate isomerase (TrpF) are sugar isomerases that are involved in histidine and tryptophan biosynthesis, respectively. Both enzymes have the (betaalpha)(8)-barrel fold and catalyze Amadori rearrangements of a thermolabile aminoaldose into the corresponding aminoketose. To identify those amino acids that are essential for catalysis, conserved residues at the active sites of both HisA and TrpF from the hyperthermophile Thermotoga maritima were replaced by site-directed mutagenesis, and the purified variants were investigated by steady-state enzyme kinetics. Aspartate 8, aspartate 127, and threonine 164 appeared to be important for the HisA reaction, whereas cysteine 7 and aspartate 126 appeared to be important for the TrpF reaction. On the basis of these results and the X-ray structure of a complex between TrpF and a bound product analogue, a reaction mechanism involving general acid-base catalysis and a Schiff base intermediate is proposed for both enzymes. A comparison of the HisA and TrpF enzymes from T. maritima and Escherichia coli showed that, at the physiological temperatures of 80 and 37 degrees C, respectively, the enzymes from the hyperthermophile have significantly higher catalytic efficiencies than the corresponding enzymes from mesophiles. These results suggest that HisA and TrpF have similar chemical reaction mechanisms and use the same strategy to prevent the loss of their thermolabile substrates.