The authors are grateful to Dr Hui Huang and Xiubao Li (South China Sea Institute of Oceanology, Chinese Academy of Sciences) for their kindness in identifying the black coral samples. “
“Archaea,

plants, and most bacteria synthesize heme using the C5 pathway, in which the first committed step is catalyzed by the enzyme glutamyl-tRNA reductase (GluTR or HemA). In Sorafenib ic50 some cases, an overproduced and purified HemA enzyme contains noncovalently bound heme. The enteric bacteria Salmonella enterica and Escherichia coli also synthesize heme by the C5 pathway, and the HemA protein in these bacteria is regulated by proteolysis. The enzyme is unstable during normal growth due to the action of Lon and ClpAP, but becomes stable when heme is limiting for growth. We describe a method for the overproduction of S. enterica HemA that yields a purified enzyme containing bound heme, identified as a b-type heme by spectroscopy. A mutant of HemA (C170A) does not contain heme when similarly purified. The mutant was used to test whether heme is directly involved in HemA regulation. When expressed from the S. selleck chemical enterica chromosome in a wild-type background, the C170A mutant allele of hemA is shown to confer an unregulated phenotype, with high levels of HemA regardless of the heme status. These results strongly

suggest that the presence of bound heme targets the HemA enzyme for degradation and is required for normal Sinomenine regulation. 5-Aminolevulinic acid (ALA) is the product of the first committed step in the heme

biosynthetic pathway, which also leads to siroheme and vitamin B12 in Salmonella enterica. Most bacteria, as well as plants and archaea, form ALA in a two-step reaction starting from the C5 skeleton of glutamate charged to glutamyl-tRNA (tRNAGlu). The initial enzyme of the pathway, glutamyl-tRNA reductase (GluTR or HemA), uses NADPH to reduce the tRNA-activated glutamate, forming GSA. GSA is subsequently converted to ALA by GSA-AT, the product of the hemL gene (reviewed in Jahn et al., 1992; Beale, 1996). The latter reaction can proceed slowly in vitro in the absence of enzyme (Hoober et al., 1988), which explains the growth of hemL mutants at about 80% of the wild-type rate in unsupplemented minimal medium (Wang et al., 1997). With ALA supplementation, hemL and hemA mutants grow as well as the wild type. We use the growth of hemL mutants in the absence or presence of ALA to study the effect of limiting the output of the heme pathway, which then reveals its regulation. 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 >10-fold increase in enzyme abundance under these conditions (Wang et al., 1999a).