title: A purified mutant HemA protein from Salmonella enterica serovar Typhimurium lacks bound heme and is defective for heme-mediated regulation in vivo.
abstract: 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 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. 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 regulation.
authors.affiliation: Department of Microbiology, Immunology and Cell Biology, West Virginia University Health Sciences Center, Morgantown, WV 26506, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Jones
authors.forename: Thomas
authors.initials: T
authors.lastname: Elliott
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006418:Heme / Q000032:analysis*; D055550:Protein Stability; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000502:physiology*; D013057:Spectrum Analysis
chemical_list: D006418:Heme; D000445:Aldehyde Oxidoreductases; C067282:glutamyl tRNA reductase