Expression and characterization of a heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Iron acquisition requires oxidative cleavage of the heme macrocycle.
A full-length heme oxygenase gene from the pathogenic bacterium Corynebacterium diphtheriae has been subcloned and expressed in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that results in the accumulation of biliverdin within the E. coli cells. The purified heme oxygenase forms a 1:1 complex with heme (Kd = 2.5 +/- 1 microM) and has hemeprotein spectra similar to those previously reported for the purified eukaryotic heme oxygenases. In the presence of an E. coli NADPH-dependent reductase isolated during the purification of Hmu O, the heme-Hmu O complex is catalytically turned over to yield biliverdin IXalpha and carbon monoxide. A number of redox partners were investigated for their ability to reconstitute Hmu O activity in vitro. Of these the most efficient appeared to be the recombinant NADH-dependent putidaredoxin/putidaredoxin reductase from Pseudomonas putida. As with the E. coli NADPH-dependent reductase the final products of the reaction were biliverdin IXalpha and carbon monoxide. This is the first bacterial heme oxygenase to be described to date. The close relationship between iron acquisition and pathogenesis suggests that the release of iron from heme by heme oxygenase may play a crucial role in the pathogenicity of C. diphtheriae.
Wilks
A
A
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA. wilks@cgl.ucsf.edu
Schmitt
M P
MP
D002384:Catalysis; D003001:Cloning, Molecular; D003353:Corynebacterium diphtheriae / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
D006418:Heme; D007501:Iron; D006419:Heme Oxygenase (Decyclizing)