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.

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…Heme-dependent degradation of GtrR is lost in mutants defective in genes encoding the Lon and ClpAP proteases. Cysteine 170 of GtrR was shown to be necessary for heme binding, and a C170A mutant protein is constitutively expressed in vivo (350).…”

Heme Synthesis and Acquisition in Bacterial Pathogens

“…The Clp and Lon proteases are responsible for this reduction in HemA levels [61]. Furthermore, mutations in HemA have been described that render HemA resistant to heme- and protease-mediated degradation, indicating that HemA binds excess heme, and holo-HemA but not apo-HemA is a substrate for proteolytic degradation [62,63]. In this manner, cellular levels of heme can regulate the first step of heme synthesis and limit the unnecessary synthesis of heme intermediates as well as the consumption of iron.…”

Functional analysis of anS-adenosylhomocysteine hydrolase homolog of chestnut blight fungus

“…The PCR product was cloned into the expression vector pET28a (Novagen, Darmstadt, Germany) to generate pET28a‐ sahh . Transformed Escherichia coli BL21 (λDE3)/pET28a‐ sahh were induced with isopropyl‐b‐ d ‐thiogalactopyranoside (IPTG), lysed, and purified by nickel affinity chromatography (detailed primer sequence, expression, and purification are described in Supporting information, Data S1; Jones & Elliott, ).…”