Crystal structure of Pseudomonas mevalonii HMG-CoA reductase at 3.0 angstrom resolution.

The rate-limiting step in cholesterol biosynthesis in mammals is catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a four-electron oxidoreductase that converts HMG-CoA to mevalonate. The crystal structure of HMG-CoA reductase from Pseudomonas mevalonii was determined at 3.0 angstrom resolution by multiple isomorphous replacement. The structure reveals a tightly bound dimer that brings together at the subunit interface the conserved residues implicated in substrate binding and catalysis. These dimers are packed about a threefold crystallographic axis, forming a hexamer with 23 point group symmetry. Difference Fourier studies reveal the binding sites for the substrates HMG-CoA and reduced or oxidized nicotinamide adenine dinucleotide [NAD(H)] and demonstrate that the active sites are at the dimer interfaces. The HMG-CoA is bound by a domain with an unusual fold, consisting of a central alpha helix surrounded by a triangular set of walls of beta sheets and alpha helices. The NAD(H) is bound by a domain characterized by an antiparallel beta structure that defines a class of dinucleotide-binding domains.

[1]  H. Narita,et al.  The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase. , 1988, The Journal of biological chemistry.

[2]  T. Osborne,et al.  Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element. , 1988, The Journal of biological chemistry.

[3]  B. C. Wang.  Resolution of phase ambiguity in macromolecular crystallography. , 1985, Methods in enzymology.

[4]  V. Rodwell,et al.  Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Site-directed mutagenesis and characterization of the mutant enzymes. , 1989, The Journal of biological chemistry.

[5]  V. Rodwell,et al.  His865 is the catalytically important histidyl residue of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase. , 1993, The Journal of biological chemistry.

[6]  G. Schulz.  Binding of nucleotides by proteins , 1992, Current Biology.

[7]  R. V. Omkumar,et al.  Phosphorylation of Ser871 impairs the function of His865 of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase. , 1994, The Journal of biological chemistry.

[8]  M G Rossmann,et al.  The locked rotation function. , 1990, Acta crystallographica. Section A, Foundations of crystallography.

[9]  J. D. Elliott,et al.  Slow binding inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. , 1990, Biochemistry.

[10]  V. Rodwell,et al.  Mevalonate utilization in Pseudomonas sp. M. Purification and characterization of an inducible 3-hydroxy-3-methylglutaryl coenzyme A reductase. , 1985, The Journal of biological chemistry.

[11]  S. S. Katiyar,et al.  Evidence for catalytic site cysteine and histidine by chemical modification of β-hydroxy-β-methylglutaryl-coenzyme a reductase , 1986 .

[12]  R. D. Simoni,et al.  The role of the membrane domain in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase. , 1992, The Journal of biological chemistry.

[13]  D. Hardie,et al.  Phosphorylation and inactivation of HMG‐CoA reductase at the AMP‐activated protein kinase site in response to fructose treatment of isolated rat hepatocytes , 1992, FEBS letters.

[14]  M. Kirtley,et al.  Some properties and mechanism of action of the beta-hydroxy-beta-methylglutaryl coenzyme A reductase of yeast. , 1967, Biochemistry.

[15]  D. Grahame Hardie,et al.  Calmodulin‐dependent multiprotein kinase and protein kinase C phosphorylate the same site on HMG‐CoA reductase as the AMP‐activated protein kinase , 1990, FEBS Letters.

[16]  Brian W. Matthews,et al.  An efficient general-purpose least-squares refinement program for macromolecular structures , 1987 .

[17]  N. Xuong,et al.  Use of the multiwire area detector diffractometer as a national resource for protein crystallography , 1985 .

[18]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[19]  V. Rodwell,et al.  Cloning, sequencing, and overexpression of mvaA, which encodes Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase , 1989, Journal of bacteriology.

[20]  I. Shechter,et al.  Studies on the catalytic site of rat liver HMG-CoA reductase: interaction with CoA-thioesters and inactivation by iodoacetamide. , 1989, Journal of lipid research.

[21]  C. Debouck,et al.  Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli. , 1988, Archives of biochemistry and biophysics.

[22]  N. Xuong,et al.  Strategy for data collection from protein crystals using a multiwire counter area detector diffractometer , 1985 .

[23]  D. Hardie,et al.  Regulation of HMG‐CoA reductase: identification of the site phosphorylated by the AMP‐activated protein kinase in vitro and in intact rat liver. , 1990, The EMBO journal.

[24]  P. Edwards,et al.  Transcriptional regulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver. , 1985, The Journal of biological chemistry.

[25]  T. Osborne,et al.  5′ end of hmg CoA reductase gene contains sequences responsible for cholesterol-mediated inhibition of transcription , 1985, Cell.

[26]  P. Edwards,et al.  Functional size of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase as determined by radiation inactivation. , 1985, The Journal of biological chemistry.

[27]  V. Rodwell,et al.  Identification of the principal catalytically important acidic residue of 3-hydroxy-3-methylglutaryl coenzyme A reductase. , 1990, The Journal of biological chemistry.

[28]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[29]  W. Dunn,et al.  Immunological evidence for eight spans in the membrane domain of 3- hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum , 1992, The Journal of cell biology.

[30]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[31]  A. Endo,et al.  The discovery and development of HMG-CoA reductase inhibitors. , 1992, Journal of lipid research.

[32]  V. Rodwell,et al.  Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Characterization and chemical modification. , 1989, The Journal of biological chemistry.

[33]  V. Rodwell,et al.  (S)-3-hydroxy-3-methylglutaryl coenzyme A reductase, a product of the mva operon of Pseudomonas mevalonii, is regulated at the transcriptional level , 1989, Journal of bacteriology.

[34]  T. A. Jones,et al.  A graphics model building and refinement system for macromolecules , 1978 .

[35]  V. Rodwell,et al.  Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. , 1992, The Journal of biological chemistry.

[36]  V. Rodwell,et al.  Transport of mevalonate by Pseudomonas sp. strain M , 1984, Journal of bacteriology.

[37]  J. Goldstein,et al.  Regulation of the mevalonate pathway , 1990, Nature.

[38]  R. Simon,et al.  The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase. , 1992, The Journal of biological chemistry.

[39]  Jones Ta,et al.  Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. , 1985, Methods in enzymology.