Expression and Characterization of a Heme Oxygenase (Hmu O) fromCorynebacterium diphtheriae

Abstract

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 (K d ‫؍‬ 2.5 ؎ 1 M) 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 IX␣ 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 NADPHdependent reductase the final products of the reaction were biliverdin IX␣ 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.

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum

“…For haem utilization, C. glutamicum, as well as its pathogenic relative Corynebacterium diphtheriae, depends on a haem uptake apparatus composed of the ABC transporter HmuTUV, several cell surface haem-binding proteins (Allen & Schmitt, 2009Drazek et al, 2000;Frunzke et al, 2011) and a haem oxygenase (HmuO), which catalyses the intracellular degradation of the tetrapyrrol ring to abiliverdin, free iron (Fe 3+ ) and carbon monoxide Schmitt, 1997;Wilks & Schmitt, 1998). Acquisition of haem, however, exposes the respective organism to the toxicity associated with high levels of haem.…”

The Heme Oxygenase(s)-Phytochrome System of Pseudomonas aeruginosa

“…In the well studied eukaryotic HOs, NADPH and NADPH-cytochrome P450 reductase serve as the electron donor (1,2). Bacterial HOs have only recently been described, and the HO from Corynebacterium diphtheriae (HmuO) was among the first ones to be discovered (3). In C. diphtheriae the HO reaction is utilized to release iron from heme under pathogenic (i.e.…”

“…In C. diphtheriae the HO reaction is utilized to release iron from heme under pathogenic (i.e. free-iron limiting) conditions (3). In fact, the breakdown of heme to mine iron is thought to be the major function of HOs from pathogenic organisms, thus allowing them to overcome the low concentrations of free-iron necessary for successful colonization (infection).…”

Bacillus subtilis Fur represses one of two paralogous haem-degrading monooxygenases

“…Members of this A. Gaballa and J. D. Helmann family have been characterized in several bacteria, including Neisseria spp. (Zhu et al, 2000), Corynebacterium diphtheriae (Wilks & Schmitt, 1998) and Pseudomonas aeruginosa (Ratliff et al, 2001). The second family of bacterial haem monooxygenases are part of the large and diverse family of Pfam03992 proteins that share an ABM motif (Puri & O'Brian, 2006;Skaar et al, , 2006Wu et al, 2005).…”

“…Haem monooxygenases bind haemin in vitro and, in the presence of a suitable reductant, haem is oxidatively degraded (Frankenberg-Dinkel, 2004;Wilks & Schmitt, 1998). HmoA and HmoB were overexpressed and purified from E. coli as His-tagged fusion proteins.…”