Editor: Jörg Simon
A purified mutant HemA protein from Salmonella enterica serovar Typhimurium lacks bound heme and is defective for heme-mediated regulation in vivo
Version of Record online: 25 MAR 2010
© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Letters
Volume 307, Issue 1, pages 41–47, June 2010
How to Cite
Jones, A. M. and Elliott, T. (2010), A purified mutant HemA protein from Salmonella enterica serovar Typhimurium lacks bound heme and is defective for heme-mediated regulation in vivo. FEMS Microbiology Letters, 307: 41–47. doi: 10.1111/j.1574-6968.2010.01967.x
- Issue online: 4 MAY 2010
- Version of Record online: 25 MAR 2010
- Received 14 December 2009; revised 3 March 2010; accepted 3 March 2010.Final version published online April 2010.
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.