Conditional Stability of the HemA Protein (Glutamyl-tRNA Reductase) Regulates Heme Biosynthesis in Salmonella typhimurium

Abstract

In many bacteria, including the enteric species Salmonella typhimurium and Escherichia coli, heme is synthesized starting from glutamate by a pathway in which the first committed step is catalyzed by the hemA gene product, glutamyl-tRNA reductase (HemA). We have demonstrated previously that when heme limitation is imposed on cultures of S. typhimurium, HemA enzyme activity is increased 10- to 25-fold. Western (immunoblot) analysis with monoclonal antibodies reactive with HemA revealed that heme limitation results in a corresponding increase in the abundance of the enzyme. Similar regulation was also observed for E. coli. The near absence of regulation of hemA-lacoperon fusions suggested a posttranscriptional control. We report here the results of pulse-labeling and immunoprecipitation studies of this regulation. The principal mechanism that contributes to elevated HemA abundance is protein stabilization. The half-life of HemA protein is ≃20 min in unrestricted cells but increases to >300 min in heme-limited cells. Similar regulation was observed for a HemA-LacZ hybrid protein containing almost all of the HemA protein (416 residues). Sodium azide prevents HemA turnover in vivo, suggesting a role for energy-dependent proteolysis. This was confirmed by the finding that HemA turnover is completely blocked in a lon clpP double mutant of E. coli. Each single mutant shows only a small effect. The ClpA chaperone, but not ClpX, is required for ClpP-dependent HemA turnover. A hybrid HemA-LacZ protein containing just 18 amino acids from HemA is also stabilized in thelon clpP double mutant, but this shorter fusion protein is not correctly regulated by heme limitation. We suggest that the 18 N-terminal amino acids of HemA may constitute a degradation tag, whose function is conditional and modified by the remainder of the protein in a heme-dependent way. Several models are discussed to explain why the turnover of HemA is promoted by Lon-ClpAP proteolysis only when sufficient heme is available.

HemR is an OmpR/PhoB‐like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes

“…hemA and hemL are thus placed at key positions to govern ALA concentrations in the cells. Regulation of the first step has been reported taking place at the protein level in bacteria, through an elegant haem-triggered feedback proteolytic mechanism (Wang et al, 1999). L. biflexa has an operon including most of the haem biosynthesis genes hemA/C/D/B/L/E/N/G (Guégan et al, 2003), a potential target for efficient transcriptional control.…”

A purified mutant HemA protein from Salmonella enterica serovar Typhimurium lacks bound heme and is defective for heme-mediated regulation in vivo

“…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, Fig.…”

“…as well as some eukaryotic enzymes, mostly expressed in E. coli, contains tightly bound heme (Vothknecht et al, 1996;. This work focuses on S. enterica, the species in which regulation by proteolysis was discovered (Wang et al, 1999a).…”

“…Regulation is characterized by a marked instability (half-life-20 min) of the HemA enzyme during normal growth. Stabilization of the protein occurs in response to heme limitation, and leads to a 4 10-fold increase in enzyme abundance under these conditions (Wang et al, 1999a). The two ATP-dependent proteases responsible for HemA degradation have been identified as Lon and ClpAP (Wang et al, 1999a).…”

“…Stabilization of the protein occurs in response to heme limitation, and leads to a 4 10-fold increase in enzyme abundance under these conditions (Wang et al, 1999a). The two ATP-dependent proteases responsible for HemA degradation have been identified as Lon and ClpAP (Wang et al, 1999a). These enzymes are not thought to be limiting when HemA accumulates, and there is no evidence for a protease adaptor acting as RssB does in the RpoS system (Bougdour et al, 2008).…”