Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem- and chlorophyll-biosynthesis pathways in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The active site possesses an unusual dipyrromethane cofactor which is extended during the reaction by the sequential addition of the four substrate molecules. The cofactor is linked covalently to the enzyme through a thioether bridge to the invariant Cys254. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. The expression of a codon-optimized gene for PBGD from Arabidopsis thaliana (thale cress) has permitted for the first time the X-ray analysis of the enzyme from a higher plant species at 1.45 Å resolution. The A. thaliana structure differs appreciably from the E. coli and human forms of the enzyme in that the active site is shielded by an extensive well defined loop region (residues 60-70) formed by highly conserved residues. This loop is completely disordered and uncharacterized in the E. coli and human PBGD structures. The new structure establishes that the dipyrromethane cofactor of the enzyme has become oxidized to the dipyrromethenone form, with both pyrrole groups approximately coplanar. Modelling of an intermediate of the elongation process into the active site suggests that the interactions observed between the two pyrrole rings of the cofactor and the active-site residues are highly specific and are most likely to represent the catalytically relevant binding mode. During the elongation cycle, it is thought that domain movements cause the bound cofactor and polypyrrole intermediates to move past the catalytic machinery in a stepwise manner, thus permitting the binding of additional substrate moieties and completion of the tetrapyrrole product. Such a model would allow the condensation reactions to be driven by the extensive interactions that are observed between the enzyme and the dipyrromethane cofactor, coupled with acid-base catalysis provided by the invariant aspartate residue Asp95.
Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å ), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Å resolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.
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