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Rupert C Wilmouth

Publications and source records attributed to Rupert C Wilmouth.

6 recordsLinked to original sources
PubMed2006

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 alpha1-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.

Amides↗
PubMed2003

Crystal structure of carbapenem synthase (CarC).

The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron- and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C2221), consistent with solution studies. CarC monomers contain a double-stranded beta-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with l-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis.

Binding Sites↗
PubMed2002

X-ray structure of a serine protease acyl-enzyme complex at 0.95-A resolution.

Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-A resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO2H at approximately pH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 A (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 A (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.

Animals↗
PubMed2002

Toward a structural understanding of the dehydratase mechanism.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.

Amino Acid Sequence↗
PubMed2002

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.

Amino Acid Sequence↗