Participation of the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase in the inactivation and degradation of the enzyme in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a (4Fe-4S) cluster which is essential for activity. The enzyme also undergoes removal of 11 N-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff et al. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t$\sb{1/2}$ = 16 min) in exponentially growing cells. The amidotransferase produced by the FeS3 integrant was stable in growing cells, but was inactivated and degraded in glucose-starved cells more rapidly (t$\sb{1/2}$ = 35 min) than the wild type enzyme. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded a rate (t$\sb{1/2}$ = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O$\sb2$ and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O$\sb2$ dependent inactivation is the rate determining step in degradation of the enzyme. Inactive amidotransferase which accumulates in vivo when wild type B. subtilis cells are treated with chloramphenicol or when a relA strain of B. subtilis is starved for amino acids was characterized. (Abstract shortened with permission of author.)

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry, Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Grandoni, Jerry Alan

Contributors dc:contributor

• Switzer, Robert L.

• Chemistry, Biochemistry

Statement dc:rights

• Copyright 1989 Grandoni, Jerry Alan

Language dc:language

eng

Identifier

AAI8924825
(UMI)AAI8924825

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/22841