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Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens.

ABSTRACT: Thermal inactivation and reactivation of pantothenate hydrolase were studied in whole cells of Pseudomonas fluorescens. The enzyme is susceptible to thermal inactivation in whole cells at 37-40 degrees C, and is reactivated when the temperature is lowered again. Chloramphenicol does not prevent reactivation. The activation energy of enzyme inactivation in vivo is about 540kJ/mol. This activation energy is 220kJ/mol in vitro, but it is increased to 550-630kJ/mol by several metabolites, such as succinate, glyoxylate and oxalate. Generally, good carbon sources, causing rapid growth, protect the enzyme from thermal inactivation in vivo, and enable reactivation to occur at a fast rate. The enzyme is also inactivated below 35 degrees C, showing an activation energy of about 35kJ/mol. Good carbon sources prevent this inactivation as well, and cause slight reactivation. Glycine, although not utilized for growth, protects the enzyme well from this inactivation but not from inactivation at 37-40 degrees C, and prevents reactivation totally. From the activation energies of inactivation and the effects of the various carbon sources, it appears possible that changes in the concentrations of intracellular metabolites may be responsible for the changes in inactivation and reactivation.

SUBMITTER: Airas RK

PROVIDER: S-EPMC1174306 | biostudies-other | 1972 Nov

REPOSITORIES: biostudies-other

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Project description:The alginate-producing bacterium Pseudomonas fluorescens utilizes the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to metabolize fructose, since the upper part of its Embden-Meyerhof-Parnas pathway is defective. Our previous study indicated that perturbation of the central carbon metabolism by diminishing glucose-6-phosphate dehydrogenase activity could lead to sugar phosphate stress when P. fluorescens was cultivated on fructose. In the present study, we demonstrate that PFLU2693, annotated as a haloacid dehalogenase-like enzyme, is a new sugar phosphate phosphatase, now designated Spp, which is able to dephosphorylate a range of phosphate substrates, including glucose 6-phosphate and fructose 6-phosphate, in vitro The effect of spp overexpression on growth and alginate production was investigated using both the wild type and several mutant strains. The results obtained suggested that sugar phosphate accumulation caused diminished growth in some of the mutant strains, since this was partially relieved by spp overexpression. On the other hand, overexpression of spp in fructose-grown alginate-producing strains negatively affected both growth and alginate production. The latter implies that Spp dephosphorylates the sugar phosphates, thus depleting the pool of these important metabolites. Deletion of the spp gene did not affect growth of the wild-type strain on fructose, but the gene could not be deleted in the alginate-producing strain. This indicates that Spp is essential for relieving the cells of sugar phosphate stress in P. fluorescens actively producing alginate. IMPORTANCE:In enteric bacteria, the sugar phosphate phosphatase YigL is known to play an important role in combating stress caused by sugar phosphate accumulation. In this study, we identified a sugar phosphate phosphatase, designated Spp, in Pseudomonas fluorescens Spp utilizes glucose 6-phosphate, fructose 6-phosphate, and ribose 5-phosphate as substrates, and overexpression of the gene had a positive effect on growth in P. fluorescens mutants experiencing sugar phosphate stress. The gene was localized downstream of gnd and zwf-2, which encode enzymes involved in the pentose phosphate and Entner-Doudoroff pathways. Genes encoding Spp homologues were identified in similar genetic contexts in some bacteria belonging to several phylogenetically diverse families, suggesting similar functions.

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Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens.

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