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 13C-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-P-gluconate 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.

FIG. 1.

Oxidative PP pathway and related reactions of B. subtilis. Relevant genes are indicated in black boxes. At least six more genes show significant similarity to gdh (35).

FIG. 2.

Maximum specific growth rates (A) and fractions of serine derived through the PP pathway (B) for wild-type B. subtilis and its isogenic zwf and zwf gntK mutants during growth on glucose. After correction for the labeling pattern in glycerol, the percentages of serine derived through the PP pathway were 32%, 8%, and 4% ± 1%, respectively. The growth rate error bars indicate the deviations based on duplicate experiments. The flux ratio error bars indicate the experimental measurement errors based on a single analysis, assuming that there was a standard error of 1% in the MS signals.

FIG. 3.

Maximum specific growth rates (A) and (glycerol-corrected) fractions of serine derived through the PP pathway (B) for wild-type B. subtilis and 6-P-gluconate dehydrogenase mutants. The growth rate error bars indicate the deviations based on duplicate experiments. The flux ratio error bars indicate the experimental measurement error based on a single analysis, assuming that there was a standard error of 1% in the MS signals. Asterisks indicate yqjI mutants that were adapted for growth on glucose alone.

FIG. 4.

NADP⁺-dependent (solid bars) and NAD⁺-dependent (open bars) 6-P-gluconate dehydrogenase activities in crude cell extracts of B. subtilis 1012 gntZ and yqjI mutants. The error bars indicate standard deviations based on triplicate experiments.

FIG. 5.

Alignment of N-terminal dinucleotide binding domains of putative 6-P-gluconate dehydrogenases. The GenBank accession numbers are indicated in parentheses. Conserved residues at positions 33 and 34 are indicated by white type on a black background. Residues conserved in at least 70% of the sequences in a class are indicated by shading. Asterisks indicate residues conserved in all three classes.