The periplasmic modifier protein for methanol dehydrogenase in the methylotrophs Methylophilus methylotrophus and Paracoccus denitrificans.

A modifier protein (M-protein), which increases the affinity of methanol dehydrogenase (MDH) for alcohols but decreases its affinity for formaldehyde, has been partially purified from Methylophilus methylotrophus and Paracoccus denitrificans. Analysis was complicated by non-protein factors in bacterial extracts that are able to mimic M-protein in one of its functions-that of increasing the activity of MDH with butane-1,3-diol in the dye-linked assay system. The 67 kDa polypeptide, previously identified as a subunit of the M-protein, is an unrelated cytoplasmic protein. The M-protein is exclusively periplasmic and is a multimeric protein with subunits of 45 kDa. The M-protein is active in the 'physiological' assay system with the specific cytochrome c electron acceptor for MDH, lowering its affinity for formaldehyde. It has its maximum effect when the ratio of M-protein:MDH is 1:5 but its concentration in the periplasm is much lower than 20% of that of MDH.     

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome cL) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome cL. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome cL, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large (Mr 23,000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamine dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome cL and to donate electrons to cytochrome cH. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.     

Aerobic and anaerobic growth of Paracoccus denitrificans on methanol

1. The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate.
2. Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme.
3. Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions.
4. Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured.
5. From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.

     

The oxidation of methanol in gram-negative bacteria

Abstract This is a brief review of the structure and interaction of methanol dehydrogenase and its electron acceptor cytochrome c _L , their regulation by modifier protein, and the synthesis and assembly of the 'methanol oxidase' system.     

Role of CO-binding cytochrome c in enzymatic oxidation of methane by the bacterium Methylococcus capsulatus

The cytochrome c spectrally related to cco cytochromes has been isolated and purified from the methane-oxidizing bacterium Methylococcus capsulatus. The cytochrome binds CO but does not bind other substrates of methane monooxygenase, does not activate the methane monooxygenase reaction and is not a component of methane monooxygenase. In the methanol dehydrogenase enzymatic system cytochrome cco functions as electron acceptor. A possible role of cytochrome cco as electron carrier intermediate in the sequence of the dehydrogenase and oxidase enzymatic systems of M. capsulatus is discussed.     

NAD-dependent, PQQ-containing methanol dehydrogenase: a bacterial dehydrogenase in a multienzyme complex

Cell-free extracts of methanol-grown Nocardia sp. 239 only show significant dye-linked methanol-oxidizing activity when NAD+ is added to the assay mixture. This activity resides in a multienzyme complex which could be resolved into 3 components, namely the methanol dehydrogenase, NAD-dependent aldehyde dehydrogenase and NADH dehydrogenase. In its dissociated form, the methanol dehydrogenase no longer shows dye reduction and although rises in the absorbance values around 340 nm are seen on addition of methanol plus NAD+ to the enzyme, this is not due to NADH production. However, dye reduction (NAD dependent) could be restored on incubating methanol dehydrogenase with the corresponding NADH dehydrogenase, obtained from the enzyme complex. It is concluded that this novel methanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogenase via a mechanism in which NAD+ and PQQ are involved.     

The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria.

The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these MDHs with their specific electron acceptor, cytochrome cL, has been studied using a novel assay system. Electrostatic reactions are involved in 'docking' of the two proteins. EDTA inhibits the reaction by a process involving neither metal chelation nor the 'docking' process. Chemical modification studies showed that the two proteins interact by a 'docking' process involving interactions of lysyl residues on MDH and carboxyl residues on cytochrome cL. When 'zero length', two stage cross-linking was done (with proteins from both bacteria), the alpha-subunits of MDH cross-linked with cytochrome cL by way of lysyl groups on MDH and carboxyl groups on the cytochrome. Tuna mitochondrial cytochrome c provided a model for cytochrome cH which is the electron acceptor for cytochrome cL in the 'methanol oxidase' electron transport chain. Tuna cytochrome c was shown to form crosslinked products with carboxyl-modified cytochrome cL. MDH and tuna cytochrome c competed for the same domain on cytochrome cL. It was concluded that MDH reacts with cytochrome cL by an electrostatic reaction which involves carboxyl groups on cytochrome cL and amino groups on the alpha-subunit of MDH. The same domain on cytochrome cL is involved in subsequent 'docking' with its electron acceptor.     

The role of the MxaD protein in the respiratory chain of Methylobacterium extorquens during growth on methanol

The largest of the gene clusters coding for proteins involved in methanol oxidation is the cluster mxaFJGIR(S)ACKLDEHB. Disruption of most of these genes leads to lack of growth on methanol. The previous results showed that the mutant lacking MxaD grows on methanol although at a low rate. This is explained by the low rate of methanol oxidation by whole cells. The specific activity of methanol dehydrogenase (MDH) is higher in the mutant but its electron acceptor (cytochrome c(L)) is unchanged. Using the purified proteins, it was shown that the rate of interaction of MDH and cytochrome c(L) was higher in the wild-type MDH containing some MxaD proteins, which was absent in the mutant MDH. It is suggested that the gene mxaD codes for the 17-kDa periplasmic protein that directly or indirectly stimulates the interaction between MDH and cytochrome c(L); its absence leads to a lower rate of respiration with methanol and therefore a lower growth rate on this substrate.     

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.     

The organisation of methanol dehydrogenase and c-type cytochromes on the respiratory membrane of Methylophilus methylotrophus

The membrane-impermeant, protein-labelling reagent [¹⁴C]isethionyl acetimidate has been used to investigate the detailed topography of the membrane-associated methanol oxidase system in the methylotrophic bacterium Methylophilus methylotrophus. The results show that methanol dehydrogenase and cytochrome c₁ are significantly less exposed on the periplasmic surface of the membrane than either cytochrome C_H or an as yet unidentified 12-kDa protein. The partial crypticity of the methanol dehydrogenase has been confirmed by fingerprinting with V8 protease. The results are discussed in terms of the electron transfer functions of the various redox proteins.     

Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus.

Acetobacter methanolicus is a unique acetic acid bacterium which has a methanol oxidase respiratory chain, as seen in methylotrophs, in addition to its ethanol oxidase respiratory chain. In this study, the relationship between methanol and ethanol oxidase respiratory chains was investigated. The organism is able to grow by oxidizing several carbon sources, including methanol, glycerol, and glucose. Cells grown on methanol exhibited a high methanol-oxidizing activity and contained large amounts of methanol dehydrogenase and soluble cytochromes c. Cells grown on glycerol showed higher oxygen uptake rate and dehydrogenase activity with ethanol but little methanol-oxidizing activity. Furthermore, two different terminal oxidases, cytochrome c and ubiquinol oxidases, have been shown to be involved in the respiratory chain; cytochrome c oxidase predominates in cells grown on methanol while ubiquinol oxidase predominates in cells grown on glycerol. Both terminal oxidases could be solubilized from the membranes and separated from each other. The cytochrome c oxidase and the ubiquinol oxidase have been shown to be a cytochrome co and a cytochrome bo, respectively. Methanol-oxidizing activity was diminished by several treatments that disrupt the integrity of the cells. The activity of the intact cells was inhibited with NaCl and/or EDTA, which disturbed the interaction between methanol dehydrogenase and cytochrome c. Ethanol-oxidizing activity in the membranes was inhibited with 2-heptyl-4-hydroxyquinoline N-oxide, which inhibited ubiquinol oxidase but not cytochrome c oxidase. Alcohol dehydrogenase has been purified from the membranes of glycerol-grown cells and shown to reduce ubiquinone-10 as well as a short side-chain homologue in detergent solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.     

Organization of Genes Required for the Oxidation of Methanol to Formaldehyde in Three Type II Methylotrophs

Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome c(L), an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome c(L) structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed.     

Characterization of Methylophaga sp. strain SK1 cytochrome cL expressed in Escherichia coli

Methylophaga sp. strain SK1 is a new restricted facultative methanol-oxidizing bacterium that was isolated from seawater. The aim of this study was to characterize the electron carriers involved in the methanol oxidation process in Methylophaga sp. strain SK1. The gene encoding cytochrome c(L) (mxaG) was cloned and the recombinant gene was expressed in Escherichia coli DH5 under strict anaerobic conditions. The recombinant cytochrome c(L) had the same molecular weight and absorption spectra as the wild-type cytochrome c(L) both in the reduced and oxidized forms. The electron flow rate from methanol dehydrogenase (MDH) to the recombinant cytochrome c(L) was similar to that from MDH to the wild-type cytochrome c(L). These results suggest that recombinant cytochrome c(L) acts as a physiological primary electron acceptor for MDH.     

Purification, characterization, and application of a novel dye-linked L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus

The distribution of dye-linked L-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked L-proline dehydrogenase (dye-L-proDH, L-proline:acceptor oxidoreductase) was found in the crude extract of some Thermococcales strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon, Thermococcus profundus DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (alpha(2) beta(2)) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the alpha-subunit (50-kDa subunit) and the beta-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-L-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70 degrees C for 10 min, and ca. 40% of the activity still remained after heating at 80 degrees C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50 degrees C for 10 min. The enzyme exclusively catalyzed L-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for L-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Delta(1)-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of L-proline at concentrations from 0.10 to 2.5 mM using the stable dye-L-proDH was achieved.     

Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth.

By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitrificans. In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.     

Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 have been characterized by complementation analysis and assigned to 10 complementation groups, Mox A1, A2, A3, and B through H (D. N. Nunn and M. E. Lidstrom, J. Bacteriol. 166:582-591, 1986). In this study we have characterized each of the mutants belonging to the 10 Mox complementation groups for the following criteria: phenazine methosulfate-dichlorophenolindophenol dye-linked methanol dehydrogenase activity; methanol-dependent whole-cell oxygen consumption; the presence or absence of methanol dehydrogenase protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the absorption spectra of purified mutant methanol dehydrogenase proteins; and the presence or absence of the soluble cytochrome c proteins of Methylobacterium sp. strain AM1, as determined by reduced-oxidized difference spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this information, we have proposed functions for each of the genes deficient in the mutants of the 10 Mox complementation groups. These proposed gene functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome cL, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome cL, three gene functions responsible for the proper association of the pyrrolo-quinoline quinone prosthetic group with the methanol dehydrogenase apoprotein, and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol.     

Characterisation of a red form of methanol dehydrogenase from the marine methylotroph Methylophaga marina

Abstract The quinoprotein methanol dehydrogenase (MDH) of the marine methylotroph Methylophaga marina is similar to that of other methylotrophs in being an α ₂ β ₂ tetramer containing two molecules of PQQ and a single atom of calcium. Its electron acceptor is cytochrome c _L and interaction of the two proteins is by way of carboxylates on the cytochrome and lysyl residues on the α subunit of MDH. The reaction was not, however, sensitive to high ionic strength as was the reaction in non-halophilic bacteria. A red form of the enzyme was sometimes produced which had a low specific activity and a low calcium content. Activity was restored by incubation with Ca²⁺ which also produced the typical (green) enzyme, with a typical absorption spectrum. This provides the first demonstration of reconstitution of active MDH from enzyme lacking calcium isolated from a wild-type methylotroph.     

PQQ glucose dehydrogenase with novel electron transfer ability

PQQ glucose dehydrogenase from Acinetobacter calcoaceticus (GDH-B) is one of the most industrially attractive enzymes, as a sensor constituent for glucose sensing, because of its high catalytic activity and insensitivity to oxygen. We attempted to engineer GDH-B to enable electron transfer to the electrode in the absence of artificial electron mediator by mimicking the domain structure of the quinohemoprotein ethanol dehydrogenase (QH-EDH) from Comamonas testosteroni, which is composed of a PQQ-containing catalytic domain and a cytochrome c domain. We genetically fused the cytochrome c domain of QH-EDH to the C-terminal of GDH-B. The constructed fusion protein showed not only intra-molecular electron transfer, between PQQ and heme of the cytochrome c domain, but also electron transfer from heme to the electrode, thereby allowing the construction of a direct electron transfer-type glucose sensor.     

UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases.

The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)