4). Cytochrome c was included as a positive control for a protein

with covalent attachment of heme, and its heme-associated Temsirolimus chemical structure peroxidase activity was detected at the expected position based on the mass of the protein (∼13 kDa), independent of treatment with thiol reagents. Both HemA1−412-His6 and HemA1−412 [C170A]-His6 were detected in Coomassie-stained gels at the predicted molecular mass of ∼46 kDa (Fig. 4, left lanes); however, peroxidase activity was only detected for the HemA1−412-His6 and only in unheated samples lacking both dithiothreitol and β-ME. Any one of three treatments, dithiothreitol, β-ME, or boiling, abolished the signal (Fig. 4, and data not shown), indicating that heme is not covalently bound. HemA1−412 [C170A]-His6 failed to produce a detectable signal under any of the conditions tested. Three bands are observed for the untreated wild-type sample. The smallest and most INCB018424 abundant band corresponds to HemA protein. The bands above it are likely aggregates as observed in other studies (Schroder et al., 1992; Verkamp et al., 1992; Schauer et al., 2002). According to one model, heme binding to HemA protein sensitizes

it to proteolytic attack. The combined observations of a regulatory defect and absence of bound heme in purified C170A led us to predict that the mutant would exhibit increased stability over wild-type HemA. Isogenic strains expressing wild-type and C170A mutant HemA in a single copy from the native locus in the S. enterica chromosome were analyzed by Western blot after inhibition of protein synthesis (Fig. 5a). Wild-type HemA was

present at lower levels than the mutants and was detectable only at the initial time point. HemA[KK], included as a positive control, remained stable over the time course of the experiment. In support of the model, the C170A mutant was nearly as stable as HemA[KK] (Fig. 5b). Our current understanding of heme biosynthesis by the C5 pathway in bacteria involves two different regulatory mechanisms: feedback inhibition of enzyme activity by heme and post-translational control of enzyme abundance by proteolysis (Wang et al., 1997; Wang et al., 1999a). Furthermore, HemA enzyme from Chlorobium vibrioforme, as well as some eukaryotic enzymes, mostly expressed in E. coli, contains tightly bound heme (Vothknecht et al., 1996; Srivastava & Beale, 2005; Srivastava et al., 2005). This work focuses on S. MycoClean Mycoplasma Removal Kit enterica, the species in which regulation by proteolysis was discovered (Wang et al., 1999a). We were unsuccessful in previous attempts to use the T7 RNA polymerase system to overexpress Salmonella HemA, which is 94% identical to the E. coli enzyme. Jahn’s group succeeded with the E. coli HemA enzyme by coexpressing the protein with the chaperones DnaJK and GrpE (Schauer et al., 2002). Analysis of the purified E. coli enzyme showed (1) no copurifying prosthetic group detectable by spectroscopy and (2) no inhibition of enzyme activity by heme in vitro. This apparent difference between the Salmonella and E.