Regulation of Glycerol Catabolism in Klebsiella aerogenes

Abstract

The utilization of glycerol as a carbon source for growth by Klebsiella aerogenes, strain 2103, involves separate aerobic (sn-glycerol-3-phosphate or G3P) and anaerobic (dihydroxyacetone or DHA) pathways of catabolism. Enzyme and transport activities of the aerobic pathway are elevated in cells grown under oxygenated conditions on glycerol or G3P. Anaerobic growth on G3P as carbon source requires the presence of an exogenous hydrogen acceptor such as fumarate; cells thus grown also are highly induced in the G3P pathway. Anaerobic growth on glycerol requires no exogenous hydrogen acceptors; cells thus grown are highly induced in the DHA pathway but almost uninduced in the G3P pathway and the addition of fumarate electron acceptors has no effect on the relative levels of the two pathways. When both glycerol and G3P are provided anaerobically with fumarate, the DHA pathway is still preferentially induced, which probably accounts for the exclusive utilization of glycerol until its exhaustion. These observations suggest the presence of a regulatory control of G3P pathway imposed by the operation of the DHA pathway. Klebsiella aerogenes, strain 1033, dissimilates glycerol by two separate pathways. In one pathway, the nutrient is phosphorylated by an adenosine 5'-triphosphate (ATP)-dependent kinase to sn-glycerol-3-phosphate (G3P). G3P is then converted to dihydroxyacetone phosphate by dehydrogenases characteristic of flavoenzymes (Fig. 1). In the other pathway, glycerol is converted to dihydroxyacetone (DHA) by an NAD-linked dehydrogenase. DHA is then phosphorylated to DHA phosphate (DHAP) by an ATP-dependent kinase. Evidence from in vivo isotopic tracer studies, as well as in vitro measurements of enzyme activities, indicates that the G3P pathway is responsible for aerobic, and the DHA pathway is responsible for anaerobic, degradation of glycerol (7, 9, 13). The presence of the latter pathway in K. aerogenes apparently permits the cell to grow anaerobically on glycerol in the absence of exogenous hydrogen acceptors, since Escherichia coli, lacking the enzymes of this pathway, is unable to do so. In the work to be described below, it is shown that K. aerogenes, like E. coli, can grow directly on G3P without prior hydrolysis, and that anaerobic growth on G3P requires exogenous hydrogen acceptors. All the enzymes of the glp regulon (a collection of operons, the expression of which is inducible by G3P) described for E.

Kinetic, dynamic, and pathway studies of glycerol metabolism byKlebsiella pneumoniae in anaerobic continuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation

“…In general, it can be stated from Figure 2A and B that the specific in vitro GDH activity (i.e., the synthesis of GDH) is not closely coupled with the glycerol uptake rate. The specific activity of GDH measured in this work is quite high compared with those reported for K. pneumoniae (Ruch et al, 1974) and other microorganisms such as Aerobacter aerogenes (Mc-Gregor et al, 1974), Hansenula ofunaensis (Yamada and Tani, 1988), and C. butyricum (Abbad-Andaloussi et al, 1996). The reported values were in the range of 0.4 to 2.4 U/mg protein, which are close to those measured for conditions of strong substrate excess in this work (Fig.…”

“…Determination of glycerol dehydrogenase (EC 1.1.1.6, glycerol:NAD + 2-oxidoreductase) activity was done as described by Ruch et al (1974). The reaction mixture (1 mL) contained 30 mM ammonium sulfate, 0.2 M glycerol, 1.2 mM NAD (adjusted to pH 7.0 with 1 M NaOH), and the eluate with enzymes in 0.1 M potassium carbonate buffer solution (pH 9.8).…”

Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p

“…Enzyme activity was inhibited by fructose-1,6-diphosphate, which acted as a noncompetitive inhibitor with Ki values of 0·14 mmol l −1 and 0·11 mmol l −1 when varying glycerol and ATP concentrations, respectively. This behaviour was also observed for other glycerol kinases (Zwaig and Lin 1966 ;Thorner and Paulus 1973 ;Ruch et al 1974).…”

Rational Co‐Immobilization of Bi‐Enzyme Cascades on Porous Supports and their Applications in Bio‐Redox Reactions with In Situ Recycling of Soluble Cofactors

“…In the case of cascade A, Gox ‐GyDH (20–200 μL) was added into a cell that contained 1 m M 2‐phenylpropionaldehyde (2 mL) and 10 m M NADPH (50 μL) in 10 m M sodium phosphate. The activities of the other dehydrogenases were measured according to modified literature procedures for Ps ‐FDH,6d Tt27‐NOX,22 Tt27‐ GDH18 and Cb ‐GyDH 23. One unit of activity was defined as the amount of enzyme that was needed to either reduce or oxidized 1 μmol of the corresponding nicotinamide cofactor at 340 nm, 25 °C, and pH 7.…”