Purification and properties of dihydroxyacetone kinase from Klebsiella pneumoniae

Abstract

Dihydroxyacetone (DHA) kinase of Klebsiella pneumoniae, a gene product of the dha regulon responsible for fermentative dissimilation of glycerol and DHA, was purified 120-fold to a final specific activity of 10 ,umol x min-I x mg of protein-' at 30°C. The enzyme, a dimer of a 53,000 + 5,000-dalton polypeptide, is highly specific for DHA (Kn, ca. 4 ,uM). Glycerol is not a substrate at 1 mM and is not an inhibitor even at 100 mM. The enzyme is not inhibited by 5 mM fructose-1,6-diphosphate. Ca2+ gives a higher enzyme activity than Mg2+ as a cationic cofactor. Escherichia coli glycerol kinase acts on both glycerol and DHA and is allosterically inhibited by fructose-1,6-diphosphate. Antibodies raised against E. coli glycerol kinase cross-reacted with K. pneumoniae glycerol kinase but not with K. pneumoniae DHA kinase.Klebsiella pneumoniae has two different systems for the dissimilation of glycerol ( Fig. 1). With molecular oxygen or nitrate available as an exogenous electron acceptor, the glp regulon is used (14). In the absence of exogenous electron acceptors, the dha regulon is used (5,9,17,23,24,28). Metabolism of glycerol through the glp system is initiated by an ATP-dependent kinase. The product, sn-glycerol 3-phosphate (G3P), is oxidized to dihydroxyacetone phosphate by one of the flavin-linked dehydrogenases. Glycerol kinase is allosterically inhibited by fructose-1,6-diphosphate which prevents excessive phosphorylation of the substrate. (It was demonstrated in Es(herichia coli that accumulation of G3P results in growth stasis [2] and overabundance of dihydroxyacetone phosphate results in the lethal production of methylglyoxal [6,12].) Metabolism of glycerol through the dim system occurs by two parallel pathways, one serving for glycerol utilization, the other for the disposal of extra hydrogen. Utilization of glycerol is catalyzed by an NADlinked dehydrogenase (16,19) which converts the substrate to dihydroxyacetone (DHA). This product is then phosphorylated by an ATP-dependent kinase. Disposal of extra hydrogen is achieved by the sequential action of a B12dependent dehydratase and an NADH-linked oxidoreductase. Glycerol is first converted to 3-hydroxypropionaldehyde which then serves as a hydrogen sink for the regeneration of NAD. The resulting 1,3-propanediol (accounting for about 50% of the glycerol consumed) is excreted into the medium (3, 4, 20, 36). E. coli differs from K. pneuimoniae is possessing only the glp system. In this study, DHA kinase was purified from K. pnelumoniae for characterization and comparison with glycerol kinases from this organism and from E. (coli.Waters Associates, Milford, Mass., and was used on a Waters gradient HPLC system. Ultrogel ACA 34 was from LKB Instruments, Inc., Rockville, Md. Blue Sepharose CL-6B and protein standards for gradient polyacrylamide gel electrophoresis were purchased from Pharmacia Fine Chemicals, Piscataway, N.J. Special enzyme-grade ammonium sulfate was obtained from Schwarz/Mann, Orangeburg, N.Y. Rabbit muscle G3P dehydrogenase was obtained from Boehr...

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon

“…Glycerol dehydrogenase activity was determined by the method of Ruch et al (24). DHA kinase activity was determined by the method of Johnson et al (12). All assays were done at 37°C.…”

“…The dha regulon is induced by dihydroxyacetone (DHA) in the absence of an exogenous electron acceptor, such as oxygen, fumarate, or nitrate (8). The enzymes of the dha regulon that are not directly involved in 1,3-PD production convert glycerol to DHA by an NAD+-dependent glycerol dehydrogenase (13,17) and then to dihydroxyacetone phosphate by an ATP-dependent DHA kinase (12); the dihydroxyacetone phosphate is further metabolized to provide carbon and energy for growth. The physiological reason for 1,3-PD formation is most likely to regenerate NAD+ needed by the DHA branch of the dha regulon (9).…”

Rei1‐like protein regulates nutritional metabolism and transport required for the asexual cycle in vitro and in vivo of a fungal insect pathogen

“…E. Hyphal biomass levels and total activities of extracellular enzymes (ECEs) and Pr1 proteases quantified from the 3-day-old CZB-BSA cultures, which contained 0.3% BSA as the sole nitrogen source for enzyme induction and 10 6 conidia ml −1 for culture initiation. sources of different sugars and polyols, including dihydroxyacetone kinase crucial for glycerol dissimilation (Johnson et al, 1984;Daniel et al, 1995), and almost all glycosyl hydrolases involved in the hydrolysis of glycosidic bonds in complex sugars, the processing of N-linked glycoproteins and the degradation of carbohydrates (Bourne et al, 2001). Moreover, 21 membrane transport protein (MFS) coding genes were also down-regulated.…”

σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

“…Interestingly, a highly expressed and conserved operon encoding a dihydroxyacetone phosphate kinase complex ( dhaKLM ; CIBE_2581-2582-2583) also appears to be controlled by σ 54 . This enzyme can be involved in glycerol metabolism, or directly in the central carbon pathway when associated to 6P-fructose aldolases (28, 29). Notably, two 6P-fructose aldolases (CIBE_0334 and CIBE_0411), one of which being well expressed in the available RNA-seq dataset (8), are annotated in the DSM 6423 genome.…”