Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response.
When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with 100 microM H2O2, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE. The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited an apparent Km of 40 mM and a Vmax of 285,000 U (mg protein)-1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases.
Terzenbach
D P
DP
Institut für Mikrobiologie, Georg August Universität, Grisebachstrasse 8, D-37 077 Göttingen, Germany.
Blaut
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002374:Catalase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000502:physiology; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000254:growth & development; D017421:Sequence Analysis; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D002374:Catalase