In vivo inactivation of glycerol dehydrogenase in Klebsiella aerogenes: properties of active and inactivated proteins

Abstract

Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation ofthe oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had similar isoelectric points (pH 4.7). Inactivation did, however, alter the gel filtration properties of the enzyme protein and, more importantly, reduced its affinity for the dye Cibacron F3GA and the coenzyme NAD+.

Physiologic Mechanisms Involved in Accumulation of 3-Hydroxypropionaldehyde during Fermentation of Glycerol by Enterobacter agglomerans

“…Provoking cessation of growth and of substrate consumption, the accumulation of this bacteriostatic compound, noticed for several enterobacterial species, probably reflects a metabolic imbalance. The biochemistry of the fermentation of glycerol has been mainly studied for the genus Klebsiella in connection with the control of the dha structural genes by a repressor (product of dhaR) and by catabolite repression and in connection with the posttranslational inactivation of Glyc DH during the switch from anaerobiosis to aerobiosis (9,13,14,18,20,24,26). Recently, Daniel and Gottschalk (11) have shown that the activity of glycerol dehydratase was growth temperature dependent since better expression of the corresponding genes was noticed at 28ЊC than at 37ЊC in a culture in which Escherichia coli expressed the genes of the dha regulon of C. freundii.…”

Purification and properties of dihydroxyacetone kinase from Klebsiella pneumoniae

“…The activity of glycerol dehydrogenase, the first enzyme of the dha pathway for carbon source utilization, is under posttranslational control: aerobic metabolism leads to irreversible inactivation of the protein by a process dependent upon the generation of some form of metabolic intermediate (15,30). The inactivation of glycerol dehydrogenase should have an indirect effect at the level of transcription when glycerol is the carbon and energy source, since DHA is the inducer of the dha regulon (4, 5).…”

Anaerobic Growth of Escherichia coli on Glycerol by Importing Genes of the dha Regulon from Klebsiella pneumoniae

“…Expression of the dha structural genes is controlled by a repressor (product of dhaR) which responds to dihydroxyacetone as the effector and by catabolite repression (Forage & Foster, 1982;Johnson et al, 1984;Ruch et al, 1974;Ruch & Lin, 1975). Control is also exerted post-translationally by the inactivation of glycerol dehydrogenase during transition from anaerobic to aerobic growth (Johnson et al, 1985;Lin et al, 1960;Ruch et al, 1980). The enzyme is destroyed irreversibly during such shifts apparently to promote induction and exploitation of the glp system ( Fig.…”

“…Oxidative inactivation of' glycerol dehydrogenase Glycerol dehydrogenase of K . pneumoniae induced anaerobically is inactivated during aerobiosis in the presence of a carbon and energy source (Lin et al, 1960;Ruch et al, 1980). It was suggested that the enzyme was attacked by oxygen radicals generated from the aerobic metabolism (Johnson et al, 1985).…”