Facultative Methylotroph Pseudomonas AM1 Research Articles

Regulation of Formaldehyde Oxidation by the Methanol Dehydrogenase Modifier Proteins of Methylophilus methylotrophus and Pseudomonas AM1

Summary: The modifier protein (M-protein) for methanol dehydrogenase (MDH) of the obligate methylotroph Methylophilus methylotrophus was purified almost to homogeneity (98% pure), characterized and shown to be similar to that previously described in the facultative methylotroph Pseudomonas AM1. It was a dimer of M r 140000, its isoelectric point was 5·6 and it showed no spectral absorbance above 320 nm. In the dye-linked MDH assay system, the M-proteins facilitated oxidation of alcohols not normally oxidized (1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol and 3-methylbutanol) by increasing the affinity of MDH for these alcohols. The effect of M-protein on the oxidation of formaldehyde was to decrease the affinity of MDH for formaldehyde by more than 97%, thus preventing any significant oxidation of formaldehyde. The M-protein also exerted its effect on the activity of MDH in the cytochrome-linked system of M. methylotrophus. The usual function of M-protein in methylotrophs is apparently to prevent formaldehyde oxidation. A dye-linked aldehyde dehydrogenase of wide specificity was partially purified and characterized; it failed to oxidize formaldehyde at a significant rate but it oxidized lactaldehyde at a rate sufficient to account for the oxidation of propanediol to lactate in whole bacteria.

The primary structures of Pseudomonas AM1 amicyanin and pseudoazurin. Two new sequence classes of blue copper proteins

The amino acid sequences of two blue copper proteins from the pink facultative methylotroph Pseudomonas AM1 (N.C.I.B. 9133) were determined. They each consist of a single polypeptide chain and bind one copper atom. Amicyanin contains 99 and pseudoazurin 123 residues. Copper-binding sites, consisting of the side chains of two histidine, one cysteine and one methionine residues, can be recognized in each protein by analogy with azurin and plastocyanin, but the spacings of the ligand residues are different, and other sequence similarity is limited. Proteins that are in the pseudoazurin sequence class can be recognized in some strains of Alcaligenes, and probably also in Paracoccus denitrificans. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50130 (23 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.

The Role of Cytochromes and Blue Copper Proteins in the Oxidation of Methanol and Methylamine in Organism 4025, an Obligate Methylotroph

Organism 4025 contained only b-type and c-type cytochromes. In the absence of any a-type cytochrome oxidase it was concluded that respiration in these bacteria is catalysed entirely by way of the o-type cytochrome oxidase. After growth on methanol, the soluble protein contained cytochromes cH and cL, and a single blue copper protein of unknown function, ‘azurin’. After growth on methylamine the soluble proteins were strikingly different. The total concentration of soluble cytochrome c was about 70% lower; cytochrome cL was about 65%, and cytochrome cH about 16% of the concentrations present after growth on methanol. Although some ‘azurin’ was still present, a second copper protein, amicyanin (94% of the total), was induced during growth on methylamine. The concentration of this protein was considerably higher than that of blue copper proteins measured in other bacteria and 35 times higher than the concentration in the facultative methylotroph Pseudomonas AM1 during growth on methylamine. It is the very high concentration of amicyanin that gave suspensions of methylamine-grown organism 4025 their blue-green colour. All of the soluble cytochrome c, and all of the methanol dehydrogenase, methylamine dehydrogenase and blue copper proteins were located in the periplasmic fraction. All these results are consistent with the conclusion that the main electron acceptor for methylamine dehydrogenase in organism 4025 is amicyanin.

The Methanol: Cytochrome c Oxidoreductase Activity of Methylotrophs

Both the soluble cytochromes c of the obligate methylotroph Methylophilus methylotrophus were rapidly autoreducible at high pH. The intramolecular autoreduction mechanism was also involved in the reduction of the cytochrome c L by methanol dehydrogenase which occurred in the absence of methanol. Pure soluble methanol dehydrogenase was shown to be able to catalyse the methanol-dependent reduction of pure cytochrome c from M. methylotrophus and from the facultative methylotroph Pseudomonas AM1 by coupling oxidation of the bacterial cytochrome to the reduction of a large excess of mammalian cytochrome c. Only one of the two cytochromes c (cytochrome c L of each organism) could react with methanol dehydrogenase to give methanol:cytochrome c oxidoreductase activity. This activity, using proteins from M. methylotrophus, was independent of pH between pH 7·0 and 9·0 and ammonia was not required. By contrast, the pH optimum for the system from Pseudomonas AM1 was 9.0 and activity was stimulated about fourfold by NH4Cl. The product of methanol oxidation was formaldehyde, which was also a substrate for the oxidoreductase system. During formaldehyde oxidation two molecules of cytochrome c were reduced for every molecule of formaldehyde oxidized. In a survey of methanol dehydrogenases and cytochromes c from Pseudomonas AM1, M. methylotrophus and the facultative autotroph Paracoccus denitrificans, it was shown that, of the two soluble cytochromes c found in each methylotroph only one was able to react with methanol dehydrogenase. The cytochrome c L from M. methylotrophus and the cytochrome c(2) of Pa. denitrificans were specific, only reacting with methanol dehydrogenase from the same organism, whereas the cytochrome c L of Pseudomonas AM1 reacted with all three methanol dehydrogenases tested.

The purification and properties of the soluble cytochromes c of the obligate methylotroph Methylophilus methylotrophus.

The obligate methylotroph Methylophilus methylotrophus contains three distinct soluble cytochromes c. The major cytochromes, cytochrome cH (about 50% of the total) and cytochrome cL (about 42%), were similar in most respects to the cytochromes cH and cL of the facultative methylotroph Pseudomonas AM1 [O'Keeffe & Anthony (1980) Biochem. J. 192, 411-419]. Cytochrome cH had a high isoelectric point, a midpoint redox potential at pH 7.0 of 373 mV and a low molecular weight (8500). The cytochrome cL had a low isoelectric point, a midpoint potential of 310 mV and a molecular weight of 21,000. The third cytochrome, cytochrome cLM, was clearly distinct from cytochromes cH and cL. Like the cytochrome cL of Pseudomonas AM1, the cytochrome cL of M. methylotrophus had the lowest midpoint potential, it reacted most rapidly with methanol dehydrogenase and it combined to the greatest extent with CO. Cytochromes cH and L of M. methylotrophus differed from those from Pseudomonas AM1 in having unusually high midpoint redox potentials for non-photosynthetic bacteria and in exhibiting a split alpha-band at low temperatures.

The Metabolism of Pyruvate by the Facultative Methylotroph Pseudomonas AM1

Thirteen mutants unable to grow on pyruvate have been isolated from the facultative methylotroph Pseudomonas AM1. Five of these lacked 2-oxoglutarate dehydrogenase and were thus obligate methylotrophs; one lacked the E1 (decarboxylase) component and the others probably lacked the E2 (transsuccinylase) component, but this could not be confirmed because transacylases could not be measured in the oxo-acid dehydrogenases of Pseudomonas AM1. Six mutants lacked pyruvate dehydrogenase (probably the E2, trans-acetylase, component) and one lacked both oxo-acid dehydrogenases. Although unable to oxidize pyruvate, one of the pyruvate dehydrogenase mutants was able to catalyse a coenzyme A-independent decarboxylation of pyruvate. The growth properties of the pyruvate dehydrogenase mutants confirmed that the loss of this enzyme is sufficient to confer on a previously facultative methylotroph the properties of a restricted facultative methyltroph similar to the hyphomicrobia. One mutant was only able to grow on C1, C2 and C3 compounds when a supplement of glyoxylate was added but was able to grow on other multicarbon compounds such as succinate. This confirms that there is a common reaction in the pathways for assimilation of C1, C2 and C3 compounds in this organism.

The Metabolism of 1,2-Propanediol by the Facultative Methylotroph Pseudomonas AM1

Pseudomonas AM1 grows on 1,2-propanediol with a mean doubling time of 7 h. The growth responses of a wide range of mutants suggest that propanediol is assimilated into cell material by way of pyruvate and acetyl-CoA. The normal enzymes for the initial metabolism of propanediol [NAD(P)+-linked dehydrogenase, propanediol oxidase and propanediol dehydratase] were absent. The initial oxidation of propanediol was catalysed by methanol dehydrogenase but only in the presence of a large molecular weight (possibly protein) stimulatory factor that seemed to change the substrate specificity of the enzyme; the product of the oxidation was lactate.

The interaction between methanol dehydrogenase and the autoreducible cytochromes c of the facultative methylotroph Pseudomonas AM1.

Cytochromes cH and cL were autoreduced at high pH (pK greater than 10) and the autoreduced cytochromes reacted with CO. The autoreduction was first-order with respect to oxidized cytochrome c and was reversible by lowering the pH. Pure methanol dehydrogenase reduced cytochrome c (in the absence of methanol) by lowering the pK for autoreduction to less than 8.5. A mechanism is proposed for the autoreduction of cytochrome c and its involvement in the reaction with methanol dehydrogenase.

Effect of growth conditions on the involvement of cytochrome c in electron transport, proton translocation and ATP synthesis in the facultative methylotroph Pseudomonas AM1

The stoicheiometry of proton translocation, the amounts of cytochromes firmly bound to membranes, and cell yields with respect to succinate and O(2) have been measured in the facultative methylotroph Pseudomonas AM1 and in the mutant lacking cytochrome c (mutant PCT76) during carbon-limited growth and carbon-excess growth. -->H(+)/O ratios during endogenous respiration of about 4 were measured in wild-type bacteria grown in carbon-excess conditions, and in the mutant in all growth conditions. During methanol- or succinate-limited growth of wild-type bacteria the -->H(+)/O ratio increased to about 6. Cell yields with respect to succinate and O(2) were higher in wild-type than in the mutant lacking cytochrome c by an amount suggesting loss in the mutant of 30% of the ATP-generating capacity of wild-type bacteria. During carbon-limited growth on methanol or succinate some cytochrome c was tightly bound to bacterial membranes, whereas none was tightly bound in bacteria grown in batch-culture or in NH(4) (+)-limited conditions. It is proposed that the role of cytochrome c in Pseudomonas AM1 depends on growth conditions and hence on the ;needs' of the bacteria. During growth in carbon-excess conditions it is only required for methanol oxidation, mediating between methanol dehydrogenase and cytochrome a/a(3). In these conditions oxidation of NADH and succinate by way of cytochrome b and cytochrome a/a(3) occurs without the mediation of cytochrome c. This is the only route for oxidation of NADH and succinate in the cytochrome c-deficient mutant in all growth conditions. During carbon-limited growth the cytochrome c becomes bound to the membrane in such a way that it can mediate between cytochromes b and a/a(3), hence becoming involved in proton translocation and ATP synthesis during NADH and succinate oxidation. An alternative possibility is that in wild-type bacteria the cytochrome c is always involved in electron transport, but that its involvement in measurable proton translocation only occurs in carbon-limited conditions.

Acetyl-CoA production and utilization during growth of the facultative methylotroph Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate.

In Pseudomonas AM1, conversion of 3-hydroxybutyrate to acetyl-CoA is mediated by an inducible 3-hydroxybutyrate dehydrogenase, an acetoacetate: succinate coenzyme A transferase (specific for succinyl-CoA) and an inducible beta-ketothiolase. Ethanol is oxidized to acetate by the same enzymes as are involved in methanol oxidation to formate. An inducible acetyl-CoA synthetase has been partially purified and characterized; it is essential for growth only on ethanol, malonate and acetate plus glyoxylate, as shown by the growth characteristics of a mutant (ICT54) lacking this enzyme. Free acetate is not involved in the assimilation of acetyl-CoA, and hydroxypyruvate reductase is not involved in the oxidation of acetyl-CoA to glyoxylate during growth on 3-hydroxybutyrate. A mutant (ICT51), lacking 'malate synthase' activity has been isolated and its characteristics indicate that this activity is normally essential for growth, of Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate, but not for growth on other substrates such as pyruvate, succinate and C1 compounds. The growth properties of a revertant (ICT51R) and of a mutant lacking malyl-CoA lyase (PCT57) indicate that an alternative route must exist for assimilation of compounds metabolized exclusively by way of acetyl-CoA.