Crystal Structure of a Statin Bound to a Class II Hydroxymethylglutaryl-CoA Reductase

School of Biological Sciences, University of Manchester, Oxford Road, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2003; 278(22):19933-8. DOI: 10.1074/jbc.M213006200
Source: PubMed


Hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the primary target in the current clinical treatment of hypercholesterolemias with specific inhibitors of the "statin" family. Statins are excellent inhibitors of the class I (human) enzyme but relatively poor inhibitors of the class II enzymes of important bacterial pathogens. To investigate the molecular basis for this difference we determined the x-ray structure of the class II Pseudomonas mevalonii HMG-CoA reductase in complex with the statin drug lovastatin. The structure shows lovastatin bound in the active site and its interactions with residues critically involved in catalysis and substrate binding. Binding of lovastatin also displaces the flap domain of the enzyme, which contains the catalytic residue His-381. Comparison with the structures of statins bound to the human enzyme revealed a similar mode of binding but marked differences in specific interactions that account for the observed differences in affinity. We suggest that these differences might be exploited to develop selective class II inhibitors for use as antibacterial agents against pathogenic microorganisms.

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    • "The pathway enzyme HMG-CoA reductase is of particular interest in this context. It exhibits significant differences in the threedimensional structure and in the sensitivity to inhibition by the active-site inhibitors known as statins (Tabernero et al. 2003). Isopentenyl diphosphate (IPP) isomerase is another important enzyme. "
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    ABSTRACT: The alarming increase and spread of resistance among emerging and re-emerging bacterial pathogens to all clinically useful antibiotics is one of the most serious public health problems of the last decade. Thus, the search for new antibacterials directed toward new targets is not only a continuous process but also, at this time, an urgent necessity. Recent advances in molecular biological technologies have significantly increased the ability to discover new antibacterial targets and quickly predict their spectrum and selectivity. The most extensively evaluated bacterial targets for drug development are: quorum sensor biosynthesis; the two component signal transduction(TCST) systems; bacteria division machinery; the shikimate pathway; isoprenoid biosynthesis and fatty acid biosynthesis.
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    • "But do such differences exist? For HMG-CoA reductase, marked structural differences characterize the class I human enzyme and the class 2 enzyme of a bacterial nonpathogen (Tabernero et al. 2003; Hedl et al. 2004). While at present the only relevant crystal structures are those of HMG-CoA reductase, coordinates are on file for both E. faecalis and human HMG- CoA synthase, and crystallization trials for E. faecalis HMG-CoA reductase have been initiated. "
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    ABSTRACT: The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.
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