Investigation of Thermolabile Variants of the Methanol and Methylamine Dehydrogenases of Methylophilus Methylotrophus and the Effect of an 11{middle dot}5 kbp Region of the Chromosome on the Stability of these Enzymes

Abstract

The thermal stabilities in vitro of the methylamine and methanol dehydrogenases of Methylophilus methylotrophus varied, depending on the conditions in which the organism was grown. Methylamine dehydrogenase activity was more stable in extracts of cells grown on methylamine plus NH,+ or on methanol plus methylamine than when methylamine provided the sole carbon and nitrogen source. In contrast, the methanol dehydrogenase activity in extracts of methylamine-grown cells was more stable than that from cells grown on medium containing methanol with either NHt or methylamine as the nitrogen source. The stability of thermolabile forms of the dehydrogenases present in extracts of mutants which have temperature-sensitive defects in the oxidation of C, compounds also varied with the growth conditions. When cosmid pAD833, which complemented these temperature-sensitive mutants, was present during growth of the wild-type strain on methylamine, the thermal stabilities of both methylamine and methanol dehydrogenases increased. However, the effects of subclones of pAD833 were complex and our results indicate that the DNA region carried on this cosmid encodes gene products which can both increase and decrease the in vitro thermal stability of enzymes involved in C, metabolism.

Characterization and Complementation of Mutants of Methylophilus Methylotrophus that have Thermolabile Forms of Proteins Involved in C1 Metabolism

“…It is curious that mutant NH-4 could grow at 42 "C on methylamine plus NH,+ and on methylamine plus methanol ; cells grown on these substrates oxidized methylamine at a greater rate than did wild-type cells and the methylamine oxidation system of these mutant cells was not thermolabile. The activity of methylamine dehydrogenase in extracts of such cells was low compared with that of the wild-type but, in contrast with the enzyme from methylamine-grown mutant cells, it had similar activities at 30 "C and 42 "C. This is consistent with our observations (Hutchinson & Goodwin, 1993) that the in vitro stability of methylamine dehydrogenase varies with the growth conditions and that the enzyme from cells grown on methylamine as sole carbon and nitrogen source is more susceptible to heat denaturation than that from cells grown on methylamine in the presence of an alternative carbon or nitrogen source. Other work has shown that it is unlikely that methylamine dehydrogenase catalyses the rate-limiting step in methylamine oxidation Hutchinson & Goodwin, 1993).…”

“…The activity of methylamine dehydrogenase in extracts of such cells was low compared with that of the wild-type but, in contrast with the enzyme from methylamine-grown mutant cells, it had similar activities at 30 "C and 42 "C. This is consistent with our observations (Hutchinson & Goodwin, 1993) that the in vitro stability of methylamine dehydrogenase varies with the growth conditions and that the enzyme from cells grown on methylamine as sole carbon and nitrogen source is more susceptible to heat denaturation than that from cells grown on methylamine in the presence of an alternative carbon or nitrogen source. Other work has shown that it is unlikely that methylamine dehydrogenase catalyses the rate-limiting step in methylamine oxidation Hutchinson & Goodwin, 1993). However, further work is required to ascertain whether the activity of methylamine dehydrogenase in mutant NH-4 grown at 42 "C on methylamine in the presence of an alternative carbon or nitrogen source is sufficient to account for the high rate at which whole cells oxidized methylamine.…”