<i>Corynebacterium glutamicum</i> Glyceraldehyde-3-Phosphate Dehydrogenase Isoforms with Opposite, ATP-Dependent Regulation

Abstract

Corynebacterium glutamicum gapA and gapB encode glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) that differ in molecular weight and activity in the presence of ATP. Comparative genome analysis revealed that GapA, the product of gapA, represented the canonical GAPDH that is highly conserved across the three major life forms. GapB, with an additional 110-residue-long sequence upstream of its GAPDH-specific domain, was homologous only to select microbial putative GAPDHs. Upon gene disruption, the initial growth rates of the wild-type, ΔgapA and ΔgapB strains on glucose (0.77, 0.00 and 0.76 h^–1, respectively), lactate (0.20, 0.18 and 0.15 h^–1, respectively), pyruvate (0.39, 0.29 and 0.20 h^–1, respectively), and acetate (0.06, 0.06 and 0.04 h^–1, respectively), implied that GapA was indispensable for growth on glucose, that GapB, but not GapA, affected early growth on acetate, and that GapB had a greater influence on growth under gluconeogenic conditions than GapA. The disruption of either gapA or gapB showed no significant effect on the transcription of any of the other gap cluster genes although it led to reduced triosephosphate isomerase (TPI) activities. Glycolytic GAPDH activity at low in vitro ATP concentrations was solely attributed to the 35.9-kDa GapA. At higher ATP concentrations, the same activity was attributed to the 51.2-kDa GapB. Both enzymes, however, exhibited similar NADP-dependent GAPDH activities at the higher ATP concentrations. In effect therefore, the GAPDH-catalyzed reaction at low ATP concentrations was irreversible, with all the glycolytic activity strictly NAD-dependent and attributed to GapA. At higher ATP concentrations, the reaction was reversible, with glycolytic activity NAD- or NADP-dependent and attributed to GapB, while gluconeogenic activity was attributable to both GapA and GapB.

Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions

“…Assays for GAPDH (Omumasaba et al, 2004), phosphoglycerate kinase (PGK) (Eikmanns, 1992), triosephosphate isomerase (TPI) (Eikmanns, 1992), PEPC (Inui et al, 1997) and LDH (Bunch et al, 1997) were performed as previously described. Malate dehydrogenase (MDH) activity was determined as described by Sridhar et al (2000), modified by using 0.1 M Tris/HCl (pH 8.0) and 0.15 mM NADH to obtain optimum enzyme activities.…”

“…Several key enzyme-encoding genes in the glycolytic and anaplerotic pathways, and the reductive arm of the TCA cycle, were more than twofold upregulated ( It has been demonstrated in previous work that in C. glutamicum, glycolysis under oxygen deprivation conditions is controlled at least in part by the intracellular NAD + /NADH ratio. This conclusion is particularly based on the observation that the gapA gene, which encodes the enzyme GAPDH, is inhibited by NADH (Inui et al, 2004a, b;Omumasaba et al, 2004). Moreover, under oxygen deprivation conditions for succinate production by C. glutamicum, the major anaplerotic reaction is driven by the ppc gene product PEPC, rather than by the pyc gene product PC (Inui et al, 2004b).…”

l -Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

“…All steps were performed at 4ºC unless otherwise stated. GapA and GapN activities were measured according to the methods described by Omumasaba et al (2004) and by Crow and Wittenberger (1979), respectively. G6PDH activity was measured according to the method described by Becker et al (2007).…”

Involvement of the LuxR-Type Transcriptional Regulator RamA in Regulation of Expression of thegapAGene, Encoding Glyceraldehyde-3-Phosphate Dehydrogenase ofCorynebacterium glutamicum

“…C. glutamicum possesses two GAPDHs, GapA and GapB. GapA is essential for glycolysis, and GapB is dispensable for glycolysis and important for gluconeogenesis (35). The susceptibility of GapA activity to the intracellular NADH/NAD ϩ ratio (8) suggests that the enzyme may well catalyze the rate-limiting step of glycolysis.…”