Although the human and Plasmodium enzymes share 42% amino acid sequence identity, several key differences suggest that PfTIM may turn out to be a potential drug target. We have identified a region which may be responsible for binding PfTIM to cytoskeletal elements or the band 3 protein of erythrocytes; attachment to the erythrocyte membrane may subsequently lead to the extracellular exposure of parts of the protein. This feature may be important in view of a recent report that patients suffering from P. falciparum malaria mount an antibody response to TIM leading to prolonged hemolysis. A second approach to drug design may be provided by the mutation of the largely conserved residue (Ser96) to phenylalanine in PfTIM. This difference may be of importance in designing specific active-site inhibitors against the enzyme. Finally, specific inhibition of PfTIM subunit assembly might be possible by targeting Cys13 at the dimer interface. The crystal structure of PfTIM provides a framework for new therapeutic leads.
Development of an ontology for the description of crystallization experiments and results is proposed.
Abstract:The crystal structure of a covalently cross-linked Lactobacillus casei thymidylate synthase has been determined at 2.8 Å resolution. The sites for mutation to achieve the bis-disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.Keywords: dimer interface; disulfide engineering; stability; thymidylate synthase Disulfide cross-links are an important stabilizing element in the structures of many globular proteins. Disulfide engineering has been advanced as an approach to stabilizing structures of proteins that lack such covalent cross-links in the native state~Sauer et al., 1986;Wells & Powers, 1986;Wetzel, 1987;Scrutton et al., 1988;Shirakawa et al., 1991;Wakarchung et al., 1994!, and the energetic contributions have been evaluated~Tamma & Privalov, 1998!. In the case of multimeric proteins, disulfide cross-linking of subunits affords mutant structures that are prevented from dissociation. Such a device permits a study of the chain unfolding process without the attendant complications of concomitant chain dissociation. We have been exploring the potential use of intersubunit disulfide crosslinks in the model proteins Lactobaccilus casei thymidylate synthase~ts!~Gokhale et al., 1994! and Plasmodium falciparum triosephosphate isomerase~Gopal et al., 1999!. In the case of ts, a mutant~T155C0E188C0C244T!, bearing two intersubunit crosslinks~155-1889, 188-1559, Fig. 1A,B! has been shown to be remarkably stabilized against thermal denaturation and chaotrope induced aggregation~Gokhale Agarwalla et al., 1996!. In this paper we describe the crystallographic characterization of the two engineered disulfide bridges and the dimer interface in the mutant enzyme. Gokhale et al.~1994! reported that the oxidized triple mutant remained soluble and retained secondary structure even at 90 8C, in contrast to the wild-type enzyme that precipitates at 52 8C. Furthermore the bis-disulfide cross-links abolished the aggregation observed for the wild-type enzyme at moderate concentrations of the chaotropes, urea, and guanidinium chloride. The Reprint requests to: M.R.N. Murthy, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; e-mail: mrn@mbu.iisc. ernet.in. Protein Science~1999!, 8:930-933. Cambridge University Press. Printed in the USA.
Enzymes play essential roles in all life processes and are used extensively in the biomedical and biotechnological fields. However, enzyme-related information is spread across multiple resources making its retrieval timeconsuming. In response to this challenge, the Enzyme Portal has been established to facilitate enzyme research, by providing a freely available hub where researchers can easily find and explore enzyme-related information. It integrates relevant enzyme data for a wide range of species from various resources such as UniProtKB, PDBe and ChEMBL. Here, we describe what type of enzyme-related data the Enzyme Portal provides, how the information is organized and, by show-casing two potential use cases, how to access and retrieve it.Abbreviations EC number, enzyme commission number; M-CSA, mechanism and catalytic site atlas; PDB, protein data bank; UniProtKB, universal protein knowledgebase.
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