The Bacillus subtilis yqjI Gene Encodes the NADP ⁺ -Dependent 6-P-Gluconate Dehydrogenase in the Pentose Phosphate Pathway

Abstract

Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13 C-labeling experiments, we demonstrated that yqjI encodes the NADP ؉ -dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD ؉ -dependent 6-Pgluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme.The carbon-rearranging transaldolase and transketolase reactions in the nonoxidative branch of the pentose phosphate (PP) pathway constitute the exclusive route for catabolism of pentoses. During growth on hexoses, the PP pathway becomes a major source of pentose phosphates for nucleotide biosynthesis and of the anabolic redox cofactor NADPH, the reducing equivalent for biosynthesis reactions (21,22). For this purpose, two consecutive NADP ϩ -dependent dehydrogenase reactions convert glucose-6-P into ribulose-5-P in the oxidative branch of the PP pathway. Because of its important role in central metabolism, the PP pathway has been investigated in great biochemical and genetic detail (18,19), and more recently it has also been investigated from a metabolic systems perspective (5, 26, 41) in the gram-negative model bacterium Escherichia coli.The PP pathway in the gram-positive model bacterium Bacillus subtilis, in contrast, has received very little attention, and most evidence has been indirectly inferred by comparison to E. coli (17). In particular, no biochemical data are available on the enzymes of the oxidative PP pathway, glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase, and there is no genetic evidence for the gene(s) encoding the 6-P-gluconate dehydrogenase. Based on sequence similarity, the distal gntZ gene of the gluconate operon was classified as a 6-P-gluconate dehydrogenase gene (38). Despite the presence of three homologues in the genome (35) and the homology-based suggestion that B...

Thermococcus kodakarensis : the key to affordable biohydrogen production

“…isozymes [204]. In general, these isozymes are similar, with the same cofactor preferences, but exceptions exist.…”

“…The other is F 420 -dependent and does not utilize NAD + or NADP + [205]. In addition, cell-free extracts of several other Mycobacterium and Nocardia species contain both F 420 -dependent and NADP + -dependent G6PDH activity [199] , and F 420 reduction in the oxPPP to adjust to the overall metabolic requirements of the cell [204]. However, the exact functional roles of these isozymes remains to be determined.…”

“…The exact effects are strongly dependent on the organism, the genetic background of the parent strain, and the environmental growth conditions [185,202,204,[218][219][220]. However, in general, G6PDH-deficient and 6PGDH-deficient prokaryotes exhibit remarkably mild phenotypes.…”

“…In contrast, prokaryotes with a functional ED pathway generally respond to 6PGDH knockout by rerouting through the ED pathway and by reversing the direction of the non-oxPPP, slightly increasing the flux through the EMP pathway, and activating malic enzyme [220,221]. Prokaryotes that lack a functional ED pathway display a similar response, but instead of rerouting the flux through the ED pathway, they increase flux through the EMP pathway [204]. Thus, in general, prokaryotes can compensate for disruption of G6PDH or 6PGDH.…”

Remodelamento metabólico de Paracoccidioides lutzii durante a privação de cobre determinado por análises proteômicas

“…Dentre as via metabólicas induzidas durante a depleção de cobre temos a via das pentoses-fosfato, ao qual é uma via alternativa para o metabolismo da glicose-6-fosfato que é usado principalmente para regenerar a oferta de uma célula de NADPH e para produzir a ribose-5-fosfato, usado na biossíntese de uma variedade de moléculas especializadas, incluindo nucleotídeos e os ácidos nucleicos (Sprenger, 1995;Zamboni et al, 2004). Estudos tem demonstrado que a atividade da glicose-6-fosfato desidrogenase é importante para a resistência às espécies reativas de oxigênio (MA et al, 1998).…”