Rupert C. Wilmouth

Structure and Mechanism of Anthocyanidin Synthase from Arabidopsis Thaliana.

Flavonoids are common colorants in plants and have long-established biomedicinal properties. Anthocyanidin synthase (ANS), a 2-oxoglutarate iron-dependent oxygenase, catalyzes the penultimate step in the biosynthesis of the anthocyanin class of flavonoids. The crystal structure of ANS reveals a multicomponent active site containing metal, cosubstrate, and two molecules of a substrate analog (dihydroquercetin). An additional structure obtained after 30 min exposure to dioxygen is consistent with the oxidation of the dihydroquercetin to quercetin and the concomitant decarboxylation of 2-oxoglutarate to succinate. Together with in vitro studies, the crystal structures suggest a mechanism for ANS-catalyzed anthocyanidin formation from the natural leucoanthocyanidin substrates involving stereoselective C-3 hydroxylation. The structure of ANS provides a template for the ubiquitous family of plant nonhaem oxygenases for future engineering and inhibition studies.

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl–enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme–product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the β-sheet of the active site.

Structural analyses on intermediates in serine protease catalysis

Although the subject of many studies, detailed structural information on aspects of the catalytic cycle of serine proteases is lacking. Crystallographic analyses were performed in which an acyl-enzyme complex, formed from elastase and a peptide, was reacted with a series of nucleophilic dipeptides. Multiple analyses led to electron density maps consistent with the formation of a tetrahedral species. In certain cases, apparent peptide bond formation at the active site was observed, and the electron density maps suggested production of a cis-amide rather than a trans-amide. Evidence for a cis-amide configuration was also observed in the noncovalent complex between elastase and an α1-antitrypsin-derived tetrapeptide. Although there are caveats on the relevance of the crystallographic data to solution catalysis, the results enable detailed proposals for the pathway of the acylation step to be made. At least in some cases, it is proposed that the alcohol of Ser-195 may preferentially attack the carbonyl of the cis-amide form of the substrate, in a stereoelectronically favored manner, to give a tetrahedral oxyanion intermediate, which undergoes N-inversion and/or C-N bond rotation to enable protonation of the leaving group nitrogen. The mechanistic proposals may have consequences for protease inhibition, in particular for the design of high energy intermediate analogues.

Purification, crystallization and preliminary X-ray diffraction of anthocyanidin synthase from Arabidopsis thaliana

Anthocyanidin synthase (ANS) from Arabidopsis thaliana is a non-haem iron(II)-dependent dioxygenase reported to catalyse the conversion of leucoanthocyanidins to anthocyanidins. Anthocyanidins are precursors of anthocyanins, which are a major family of pigments in higher plants. ANS was crystallized by the vapour-diffusion method using polyethylene glycol as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.0, b = 73.2, c = 87.0 A, and diffract to 2.4 A using Cu Kalpha radiation.

5,5-Fused thiophene γ-lactams as templates for serine protease inhibition

Novel 5,5-fused thiophene lactams are potent inhibitors and acylating agents of HNE and PPE.

Reaction of Clavams with Elastase Reveals a General Method for Inhibiting ‘Serine’ Enzymes

Abstract Ester derivatives of clavulanic acid acylate Ser-195 of the serine protease porcine pancreatic elastase to form stable malonyl semi-aldehyde derivatives, analogous to those formed in the inhibition of β-lactamases by clavulanic acid itself. Formation of such derivatives maybe a general way of inhibiting ‘serine’ enzymes.

β-sultams - a novel class of serine protease inhibitors

N-Benzoyl β-sultam is an irreversible inactivator of elastase by sulfonation of the active site serine.