Dr Henry Miziorko
Research and Selected Publications

Research Interests

Dr. Miziorko's research group uses chemical, biophysical, and molecular biology approaches to elucidate the structure/function relationships that account for enzyme regulation or catalysis. Currently under investigation are several enzymes important to lipid or carbohydrate metabolism including: HMG-CoA synthase, HMG-CoA lyase, phosphoribulokinase, and mevalonate kinase. 

One established project involves enzymes important to the biosynthesis of isoprenoids and sterols and related steps in ketogenesis. The active site of HMG-CoA synthase has been identified and ongoing studies are aimed at elucidating the structural basis for substrate binding and catalytic events. Directed mutagenesis, protein engineering, and physical biochemistry (ESR, ¹³C NMR spectroscopy) techniques are used to support the active site mapping work; this approach has already resulted in elucidation of the initial structure-function relationships that will account for this key biosynthetic reaction. A similar approach is used in related ongoing work that aims at identifying the catalytic and regulatory apparatus of HMG-CoA lyase. These regions have been identified by protein chemistry methods and progress is also being made in mapping point mutations that account for human HMG-CoA lyase deficiencies. The structure/function correlations for amino acids in key regions of this enzyme will be elucidated by the combined use of directed mutagenesis and mechanistic enzymology approaches. 

Recently, we have extended the strategy and approaches outlined above to an investigation of mevalonate kinase, another enzyme involved in lipid biosynthesis and a target newly implicated in inherited metabolic diseases such as mevalonic aciduria and periodic fever syndrome. Recombinant forms of the rat and human enzymes have been developed and are being exploited as we extend our initial studies, which have implicated elements of the catalytic apparatus and the ATP binding site. In addition to our current solution structure work on mevalonate kinase, our production of diffraction quality crystals of this enzyme has expedited the pursuit of X-ray diffraction studies. The crystal structure is pursued in collaboration with a departmental colleague, Prof. J.J. Kim. 

An established project involving photosynthetic carbon assimilation is currently focused on phosphoribulokinase. Structurally heterologous enzymes that catalyze the phosphorylation of ribulose 5-phosphate (a reaction unique to the Calvin cycle) are available from the usual plant sources as well as from recombinant bacterial strains. Mechanistic and structural studies on these heterologous proteins and on variants produced by directed mutagenesis have led to identification of domains involved in catalysis as well as in regulation of activity. In addition to our solution state work on a recombinant form of the R. sphaeroides enzyme, we have crystallized this protein. X-ray diffraction studies on phosphoribulokinase have been pursued in our department in collaboration with Dr. David Harrison. We recently published the first high resolution (2.5 Å) structure of this enzyme. Availability of this information has expedited design of a new generation of protein engineering experiments aimed at generating functional assignments for amino acids implicated as part of the catalytic and regulatory sites.

Selected Publications

"Inactivation of 3-Hydroxy-3-methylglutaryl-CoA Synthase and Other Acyl-CoA Utilizing Enzymes by 3-Oxobutylsulfoxyl-CoA", H.A. Charlier, C. Narasimhan, and H.M. Miziorko. Biochemistry, 36, 1551-1558 (1997).

"Evidence Supporting a Role for Histidine-235 in Cation Binding to Human 3-Hydroxy-3-methyglutaryl-CoA Lyase", J.R. Roberts and H.M. Miziorko. Biochemistry, 36, 7594-7600 (1997).

"Identification of Catalytic Residues in Human Mevalonate Kinase", D. Potter and H.M. Miziorko. Journal of Biological Chemistry, 272, 25449-25454 (1997).

"Functional Evaluation of Invariant Arginines Situated in the Mobile Lid Domain of Phosphoribulokinase", J.A. Runquist, D.H.T. Harrison, and H.M. Miziorko. Biochemistry, 37, 1221-1226 (1998).

"The Crystal Structure of Phosphoribulokinase from Rhodobacter sphaeroides Reveals a Fold Similar to Adenylate Kinase", D.H.T. Harrison, J.A. Runquist, A. Holub, and H.M. Miziorko. Biochemistry, 37, 5074-5085 (1998).

"Detection of Acetyl-S-Enzyme Reaction Intermediates of Hydroxymethylglutaryl-CoA Synthase and ß-Ketothiolase by ¹³C NMR" D. Vinarov, C. Narasimhan, and H.M. Miziorko. J. Am. Chem. Soc., 121 ,270-271 (1999).

"R. sphaeroides Phosphoribulokinase: Identification of Lysine-165 as a Catalytic Residue and Evaluation of the Contribution of Invariant Basic Amino Acids to Ribulose 5-Phosphate Binding" J.A. Runquist, D.H.T. Harrison, and H.M. Miziorko. Biochemistry, 38, 13999-14005 (1999).

"The Identification of the Allosteric Regulatory Site in Bacterial Phosphoribulokinase" G. Kung, J.A. Runquist, H.M. Miziorko, and D.H.T. Harrison. Biochemistry, 38, 15157-15165 (1999).

"Phosphoribulokinase: current perspectives on the structure/function basis for regulation and catalysis." H.M. Miziorko. Advances in Enzymology, 74, 95-127 (2000).

"HMG-CoA Synthase Reaction Intermediates: Detection of a Covalent Tetrahedral Adduct by Differential Isotope Shift ¹³C NMR Spectroscopy" D.A. Vinarov and H.M. Miziorko. Biochemistry, 39, 3360-3368 (2000).

"3-Hydroxy-3-methylglutaryl-CoA Synthase: A Role for Glutamate-95 in General Acid/Base Catalysis of C-C Bond Formation" K.Y. Chun, D.A. Vinarov, J. Zajicek, and H.M. Miziorko. J. Biol. Chem., 275, 17946-17953 (2000).

"3-Hydroxy-3-methylglutaryl-CoA Synthase: Participation of Invariant Acidic Residues in Formation of the Acetyl-S-Enzyme Reaction Intermediate" K.Y. Chun, D.A. Vinarov, and H.M. Miziorko. Biochemistry, 39,14670-14681 (2000).

"Investigation of Invariant Serine/Threonine Residues in Mevalonate Kinase: Tests of the Functional Significance of a Proposed Substrate Binding Motif and a Site Implicated in Human Inherited Disease" Y. K. Cho, S.E. Rios, J. J. P. Kim, and H.M. Miziorko. J. Biol.Chem., 276, 12573-12578 (2001).

"Functional Evaluation of Serine/Threonine Residues in the P-Loop ofR. Sphaeroides Phosphoribulokinase" J. A. Runquist, S. E. Rios,D. A. Vinarov, and H.M. Miziorko. Biochemistry, 40, 14530-14537 (2001).

"The Structure of a Binary Complex between a Mammalian Mevalonate Kinase and ATP: Insights into the Reaction Mechanism and Human Inherited Disease." Z. Fu, M. Wang, D. Potter, H.M. Miziorko, and J. J. P. Kim. J. Biol. Chem., 277, 18134-18142 (2002).

"Detection of a Covalent Tetrahedral Adduct by Differential Shift 13C NMR: Acetyl-Enzyme Reaction Intermediate Formed by 3-Hydroxy-3-methylglutaryl-CoA Synthase." H.M. Miziorko and D. A. Vinarov. Methods Enzymol. 354, 208-223 (2002).

"The Influence of Conserved Aromatic Residues in 3-Hydroxy-3-methylglutaryl-CoA Synthase." I. Misra, C.Z. Wang, and H.M. Miziorko. J. Biol. Chem., 278, 26443-26449 (2003).

"Investigation of Conserved Acidic Residues in 3-Hydroxy-3-methylglutaryl-CoA Lyase: Implications for Human Disease and for Functional Roles in a Family of Related Proteins." R.L. Tuinstra and H.M. Miziorko. J. Biol. Chem. 278, 37092-37098 (2003).

Last modified on: Wednesday, 24-Mar-2004 09:21:18 CST