Show simple item record

Files in this item

Thumbnail

Item metadata

dc.contributor.authorSgraja, Tanja
dc.contributor.authorSmith, Terry K
dc.contributor.authorHunter, William N.
dc.date.accessioned2014-04-28T14:01:01Z
dc.date.available2014-04-28T14:01:01Z
dc.date.issued2007-03-30
dc.identifier.citationSgraja , T , Smith , T K & Hunter , W N 2007 , ' Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase ' , BMC Structural Biology , vol. 7 , 20 . https://doi.org/10.1186/1472-6807-7-20en
dc.identifier.otherPURE: 3439607
dc.identifier.otherPURE UUID: f9054171-1427-48a9-af0c-505939b38de3
dc.identifier.otherWOS: 000245723000002
dc.identifier.otherScopus: 34247137519
dc.identifier.urihttps://hdl.handle.net/10023/4633
dc.descriptionThis research was supported by the German Academic Exchange Service (DAAD), the Wellcome Trust (TKS and WNH as Trust Senior Research fellows), the Biotechnology and Biological Science Research Council (Structural Proteomics of Rational Targets) and the European Synchrotron Radiation Facility.en
dc.description.abstractBackground: Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components. Results: Putative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)-over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of ( R)mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha 2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding. Conclusion: Our new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.
dc.format.extent16
dc.language.isoeng
dc.relation.ispartofBMC Structural Biologyen
dc.rights© 2007 Sgraja et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citeden
dc.subjectHuman inherited diseaseen
dc.subjectMethanococcus-jannaschiien
dc.subjectBiosynthetic-pathwayen
dc.subjectTrypanosoma-bruceien
dc.subjectCrystal-structureen
dc.subjectExpressionen
dc.subjectPurificationen
dc.subjectSuperfamilyen
dc.subjectIsoprenoidsen
dc.subjectMechanismen
dc.subjectQH301 Biologyen
dc.subjectSDG 3 - Good Health and Well-beingen
dc.subject.lccQH301en
dc.titleStructure, substrate recognition and reactivity of Leishmania major mevalonate kinaseen
dc.typeJournal articleen
dc.description.versionPublisher PDFen
dc.contributor.institutionUniversity of St Andrews. School of Biologyen
dc.contributor.institutionUniversity of St Andrews. Biomedical Sciences Research Complexen
dc.identifier.doihttps://doi.org/10.1186/1472-6807-7-20
dc.description.statusPeer revieweden
dc.identifier.urlhttp://www.scopus.com/inward/record.url?scp=34247137519&partnerID=8YFLogxKen


This item appears in the following Collection(s)

Show simple item record