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Volume 138, Issue 10

Isolation and phenotypic characterization of methanol oxidation mutants of the restricted facultative methylotroph Free

Abstract

Methanol oxidation in appears to be organized in a way similar to that in other Gram-negative methylotrophs, involving a quinoprotein methanol dehydrogenase (MDH; EC 1.1.99.8) and two soluble cytochromes. The MDH is composed of two different subunits, most probably arranged in an αβ structure. The two cytochromes are of the -type and differ in size (molecular mass 19.5 and 12.5 kDa) and isoelectric point (pI 4.6 and 9.2). The one with the lowest isoelectric point, commonly designated as cytochrome , is able to oxidize reduced MDH. Taking advantage of the ability of , a restricted facultative methylotroph, to utilize fructose as a growth substrate, mutants impaired in methanol utilization were isolated after application of optimal concentrations of ethylmethane sulphonate. Three classes of methanol oxidation mutants were obtained. Class I mutants were affected in a global regulation of the synthesis of both apo-methanol-dehydrogenase and cytochrome as well as PQQ (pyrroloquinoline quinone). Class II mutants did not produce an active MDH, but instead a comparable amount of a 65 kDa protein was found in the cell-free extract upon SDS-PAGE. This mutant protein was purified and compared to wild-type MDH. It was located in the periplasm, but unlike MDH it was composed of only two identical large subunits. Each of these subunits was able to bind one molecule of PQQ. An antiserum raised against wild-type MDH did not react with the mutant protein. Conversely, an antiserum raised against mutant protein weakly cross-reacted with wild-type MDH, suggesting that the presence of the β-subunit in MDH dramatically changes its immunochemical behaviour. Three of the class II mutants did not produce PQQ. In the presence of PQQ, partial revertants able to grow on methanol medium were obtained (class III). Class III mutants produced a stable apo-MDH consisting of α- and β-subunits and showing a normal reaction with the anti-MDH serum. Although this apo-MDH can bind PQQ, enzymic activity was not restored . This suggests that, in addition to apo-MDH and PQQ, other factors are required for the assembly of an enzymically active MDH.

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© Society for General Microbiology, 1992
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1992-10-01
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Isolation and phenotypic characterization of methanol oxidation mutants of the restricted facultative methylotroph Methylophaga marina
Microbiology 138, 2113 (1992); https://doi.org/10.1099/00221287-138-10-2113
/content/journal/micro/10.1099/00221287-138-10-2113
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