Pure methanol dehydrogenase of Pseudomonas AMI oxidizes propanediol in a transitory fashion when measured in a dye-linked assay with phenazine ethosulphate (PES) as electron acceptor. It was shown that the transitory nature of this oxidation is not because the product (lactaldehyde) is an inhibitor but because the dehydrogenase is inactivated by PES. Substrates having low affinities for the enzyme, such as propanediol, are unable to protect against this inactivation. A ‘stimulatory factor’, previously shown to facilitate the continuous oxidation by methanol dehydrogenase of propanediol to lactate, was shown to consist of two proteins, a modifier protein (M-protein) and an aldehyde dehydrogenase. The M-protein increased the affinity of methanol dehydrogenase for propanediol; this enabled the substrate to protect the enzyme against inactivation by PES, and it facilitated the continuous oxidation of low concentrations of propanediol to lactaldehyde. The M-protein was purified almost to homogeneity (more than 95% pure) and shown to be an acidic, dimeric protein having subunits of molecular weight 64000. The second protein component of the ‘stimulatory factor’ was a dye-linked aldehyde dehydrogenase which oxidized lactaldehyde to lactate but which had a greater affinity for longer-chain normal aliphatic aldehydes and the following characteristics: pH optimum, 7-0; isoelectric point, 4 0; molecular weight, 115000; and Km for lactaldehyde, 16 mm.
Beardmore-GrayM„.,
O’KeeffeD. T.,
AnthonyC.1983; The methanol: cytochrome c oxidoreduc-tase activity of methylotrophs. Journal of General Microbiology 129:923–933
BergmeyerH. U.1974; Assay of L-lactate using lactate dehydrogenase. In Methods of Enzymatic Analysis pp. 1464–1468 Edited by
Bergmeyer.H. U.
London: Academic Press;
BolbotJ. A.,
AnthonyC.1980; The metabolism of 1,2-propanediol by the facultative methylotroph Pseudomonas AMI. Journal of General Microbiology 120:245–254
BradfordM.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
DuineJ. A.,
FrankJ.1980; Studies on methanol dehydrogenase from Hyphomicrobium X. Isolation of an oxidised form of the enzyme. Biochemical Journal 187:213–219
GhoshR.,
QuayleJ. R.1979; Phenazine ethosulphate as a preferred electron acceptor to phenazine methosulphate in dye linked enzyme assays. Analytical Biochemistry 99:112–117
HoughL.,
JonesJ. K. N.1952; The synthesis of sugars from simpler substances. Part 4. Enzymic synthesis of 6-deoxy-D-fructose and 6-deoxy-L-sor-bose. Journal of the Chemical Society4052–4055
KohlerJ.,
SchwartzA. C.1982; Oxidation of aromatic aldehydes and aliphatic secondary alcohols by Hyphomicrobium spp. Canadian Journal of Microbiology 28:65–72
LaasT.,
Fast-JohanssonA.1979; Isoelectric focusing with Pharmalyte in gel rods. In Protides of the Biological Fluids 27 pp. 693–697 Edited by
Peeters.H.
Oxford: Pergamon Press;
MarisonI. W.,
AttwoodM. M.1980; Partial purification and characterization of a dye-linked formaldehyde dehydrogenase from Hyphomicrobium X. Journal of General Microbiology 117:305–313
O’KeeffeD. T.,
AnthonyC.1980; The interaction between methanol dehydrogenase and the autoreducible cytochromes c of the facultative methylotroph Pseudomonas AMI. Biochemical Journal 190:481–484