title: Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: characterization of the isolated recombinant protein and investigation of the enzyme's cation requirements.
abstract: Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase has been expressed in an active form in Escherichia coli and purified to homogeneity. Enzyme activity in crude extracts is 30-fold higher than reported for a homologous expression system. After Q-Sepharose fast-flow anion-exchange chromatography, the enzyme, which represents the first homogeneous preparation of a prokaryotic form of the protein, exhibits a specific activity of 70 units/mg. The purified enzyme is stable when stored in 20% glycerol at -80 degrees C. The recombinant bacterial enzyme cross reacts with antiserum produced against avian liver lyase, indicating some sequence homology between the two proteins. The enzyme exhibits a Km = 20 microM for (S)-HMG-CoA. Divalent cations (Mg2+ and Mn2+) markedly stimulate the enzyme activity under assay conditions; activity is only modestly increased by exogenous mercaptans. The activator constant, K(a), for Mg2+ (6.9 mM) is 3 orders of magnitude greater than that for Mn2+ (2.0 microM). While EDTA does not affect activity, o-phenanthroline treatment markedly inhibits the enzyme. In contrast, m-phenanthroline is ineffective, suggesting that the ortho isomer's effect is attributable to chelation of a tightly bound metal ion. Atomic absorption and EPR analyses of isolated enzyme indicate the presence of tightly bound copper. In enzyme expressed using standard LB broth, copper is detected at stoichiometries of only 0.07-0.10. When the growth medium is supplemented with 1 mM CuSO4, stoichiometry of copper binding increases to over 0.7 per enzyme subunit. Copper-enriched lyase displays enhanced thermal stability in comparison with enzyme that is low in metal content.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
authors.forename: C
authors.initials: C
authors.lastname: Narasimhan
authors.forename: H M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D015153:Blotting, Western; D002413:Cations, Divalent; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008670:Metals / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013054:Spectrophotometry, Atomic
chemical_list: D002413:Cations, Divalent; D008670:Metals; D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase