Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

Abstract

The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg 2؉ ions; equimolar concentrations of Mn 2؉ ions were a poor substitute for Mg 2؉ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K m of PrpB for 2-MIC was measured at 19 M, with a k cat of 105 s ؊1 . Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB K121A , PrpB H125A , and PrpB R122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k cat for 2-MIC lyase activity, respectively. The PrpB D58A and PrpB C123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg 2؉ ion.Propionate catabolism in Salmonella enterica serovar Typhimurium LT2 (herein referred to as serovar Typhimurium) occurs via the 2-methylcitric acid (2-MC) cycle (Fig. 1) (18). The 2-MC cycle of propionate metabolism was first identified in the yeast Yarrowia lipolytica and several years later was found to exist in prokaryotes (8,18,36,37). The prokaryotic enzymes of this pathway have been characterized. PrpE is the propionyl coenzyme A synthetase (14, 17), PrpC is the 2-MC synthase (18, 39), PrpD is the 2-MC dehydratase (8, 15), and PrpB is the 2-methylisocitrate (2-MIC) lyase (7,18). This work focuses on the serovar Typhimurium PrpB enzyme, which catalyzes the cleavage of 2-MIC into pyruvate and succinate (15).Comparisons of the amino acid sequence of the PrpB protein to other protein sequences identified it as a homolog of carboxyphosphonoenolpyruvate (CPEP) mutase, phosphoenolpyruvate (PEP) mutase, and isocitrate lyases (ICL) ...

TheacnDGenes ofShewenella oneidensisandVibrio choleraeEncode a New Fe/S-Dependent 2-Methylcitrate Dehydratase Enzyme That RequiresprpFFunction In Vivo

“…[2-13 C]MC was synthesized in vitro as described (17). Reactivated AcnD was added to this reaction, incubated for 1 h, and analyzed by 13 C-NMR spectroscopy. Peak assignments were based on those identified previously (17).…”

“…Glycerol was present in all protein samples and observed in the spectra due to the natural abundance of 13 C (17). 13 C-nuclear magnetic resonance (NMR) spectra were obtained at the National Magnetic Resonance Facility at the University of Wisconsin-Madison by means of a Bruker Instruments DMX-400 Avance console with a 9.4-T wide-bore magnet at 100.6 MHz.…”

“…Reaction mixtures (0.5 ml) contained [2-13 C]MC, H 6 PrpD, PrpF, or reactivated AcnD (25 g each) or PrpF and reactivated AcnD (25 g each). The reaction mixtures were allowed to incubate for 1 h at 37°C and were prepared for 13 C-NMR analysis as described above.…”

“…1A) as follows: prpB encodes 2-methylisocitrate (2-MIC) lyase (13,14,17), prpC encodes 2-MC synthase (17,18,35), prpD encodes 2-MC dehydratase (9,17), and prpE encodes propionyl-coenzyme A synthetase (19). However, other prp operons have a gene organization that differs greatly from that of these two enterics (8,17) (Fig.…”

Mutation of Phosphotransacetylase but Not Isocitrate Lyase Reduces the Virulence of Salmonella enterica Serovar Typhimurium in Mice

“…In S. enterica expression of the 1,2-propanediol utilization (pdu) genes has been linked by in vivo expression technology studies to reduced fitness during infection (4). As a control for the inability to utilize propionate, we mutated prpB, which encodes the 2-methylisocitrate lyase that catalyzes the conversion of 2-methylisocitrate to pyruvate and succinic acid, a necessary step in the metabolism of propionic acid (6,8). We were also prompted by the recently established connection between short-chain fatty acid catabolism and sirtuin (19) to investigate the role of cobB (sirtuin protein deacetylase [20]) in virulence.…”