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

Day, Darren J.; Nunn, D.; Anthony, Christopher

doi:10.1099/00221287-136-1-181

Cited by 17 publications

(17 citation statements)

References 29 publications

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“…Data from the literature suggest that a cytochrome c may be involved in accepting electrons from amicyanin (20,26), but in M. extorquens AM1, no amicyanin-specific cytochrome has been identified. However, a cytochrome c of unknown function, cytochrome c-553, has been identified in M. extorquens AM1 (16) and is therefore a possible candidate for this role. We were interested in determining whether the genes of unknown function in the methylamine utilization cluster described here encode cytochrome c-553, which is approximately 23 kDa in molecular mass.…”

Section: Resultsmentioning

confidence: 99%

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

1991

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An isolated 5.2-kb fragment of Methylobacterium extorquens AM1 DNA was found to contain a gene cluster involved in methylamine utilization. Analysis of polypeptides synthesized in an Escherichia coli T7 expression system showed that five genes were present. Two of the genes encoded the large and small subunits of methylamine dehydrogenase, and a third encoded amicyanin, the presumed electron acceptor for methylamine dehydrogenase, but the function of the other two genes is not known. The order on the 5.2-kb fragment was found to be large-subunit gene, the two genes of unknown function, small-subunit gene, amicyanin gene. The gene for azurin, another possible electron acceptor in methylamine oxidation, does not appear to be present within this cluster of methylamine utilization genes.Gram-negative bacteria of diverse systematic groups are able to utilize methylamine as a carbon, energy, and nitrogen source. These groups include facultative autotrophs such as Paracoccus denitrificans (25) (45,56), and all nonpigmented "Pseudomonas" strains (5,7,24,32).Methylobacterium extorquens AM1 utilizes methylamine via MADH (18). The enzyme consists of two large and two small (14-kDa) subunits (51), with a cofactor covalently bound to the small subunit. MADHs from other methylotrophic bacteria have a similar structure (23, 27, 42, 51), except the MADH from Methylobacillus flagellatum KT, which is a heterodimer (30). Amino acid (28) and nucleotide (14) sequences of the small subunit of MADH from M. extorquens AM1 have been published. Recent evidence has shown that the MADH cofactor has a unique structure, having two tryptophans within the polypeptide chain that are modified and covalently linked (14). A novel structure has been proposed for this cofactor (43).The copper-containing protein amicyanin is proposed to be the natural electron acceptor for MADH (17,25,34,35,54,55,57,58 Another copper-containing protein, azurin, has been found in M. extorquens AML. It accepts electrons in vitro from amicyanin, cytochrome CL, or cytochrome CH and transfers them to a terminal oxidase (54). However, it is induced only in the presence of high concentrations of copper in the growth medium. Azurin is also induced by high copper concentrations in M. flagellatum KT (17), "Methylomonas" strain J (54), and P. denitrificans (40).Although the first step of methylamine oxidation has been well studied biochemically, little is known about the organization of genes involved in methylamine metabolism. Recently, we reported the cloning and sequencing of the genes for the small subunit of MADH (14)

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Section: Resultsmentioning

confidence: 99%

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

1991

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“…However, a moxD mutant of M. extorquens AM1, which contains no methanol dehydrogenase protein, forms large amounts of that c553-type cytochrome (15). Its molecular mass of 23 kDa and its ability to bind CO, equal to that of cytochrome C553; in P. denitrificans (CO binding not shown here), suggest similarity between these c-type cytochromes in both organisms.…”

Section: Methodsmentioning

confidence: 99%

“…The physiological function of cytochrome C553 in M. extorquens AM1 is unknown at the moment. In vitro experiments show that this cytochrome mediates electrons between the cytochromes CL and CH (15), but the physiological relevance of this phenomenon is unknown.…”

Section: Methodsmentioning

confidence: 99%

Isolation, sequencing, and mutagenesis of the gene encoding cytochrome c553i of Paracoccus denitrificans and characterization of the mutant strain

et al. 1991

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The periplasmically located cytochrome c5531 of Paracoccus denitrificans was purified from cells grown aerobically on choline as the carbon source. The purified protein was digested with trypsin to obtain several protein fragments. The N-terminal regions of these fragments were sequenced. On the basis of one of these sequences, a mix of 17-mer oligonucleotides was synthesized. By using this mix as a probe, the structural gene encoding cytochrome c553, (cycB) was isolated. The nucleotide sequence of this gene was determined from a genomic bank. The N-terminal region of the deduced amino acid sequence showed characteristics of a signal sequence. Based on the deduced amino acid sequence of the mature protein, the calculated molecular weight is 22,427. The gene encoding cytochrome c553. was mutated by insertion of a kanamycin resistance gene. As a consequence of the mutation, cytochrome c553, was absent from the periplasmic protein fraction. The mutation in cycB resulted in a decreased maximum specific growth rate on methanol, while the molecular growth yield was not affected. Growth on methylamine or succinate was not affected at all. Upstream of cycB the 3' part of an open reading frame (ORF1) was identified. The deduced amino acid sequence of this part of ORF1 showed homology with methanol dehydrogenases from P. denitrificans and Methylobacterium extorquens AM1. In addition, it showed homology with other quinoproteins like alcohol dehydrogenase from Acetobacter acedi and glucose dehydrogenase from both Acinetobacter calcoaceticus and Escherichia coli. Immediately downstream from cycB, the 5' part of another open reading frame (ORF2) was found. The deduced amino acid sequence of this part of ORF2 showed homology with the moxJ gene products from P. denitficans and M. extorquens AM1.

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“…The method used was that of BeardmoreGray et al (1983). except for determination of kinetic constants for cytochrome cL, when the more rapid assay of Day & Anthony (1990) was used.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the periplasmic cytochromes c of Paracoccus denitrificans: identification of the electron acceptor for methanol dehydrogenase, and description of a novel cytochrome c heterodimer

Long

Anthony

1991

Microbiology

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This paper describes periplasmic c-type cytochromes from two strains of Paracoccus denifr@cans NCIB 8944 grown in heterotrophic or methylotrophic conditions. It is shown that the functions of two monomeric, monohaem cytochromes induced during growth on methanol have been wrongly designated in previous work. The CO-reactive cytochrome c553 (30 kDa) is not the electron acceptor for methanol dehydrogenase; this is shown to be the role of the cytochrome C552 (22 kDa). The monomeric 45 kDa cytochrome induced in conditions of oxygen insufficiency is a dihaem c-type cytochrome and does not contain haem b as previously assumed. In addition to these cytochromes, the Oxford strain of NCIB 8944 contains two cytochrome c complexes. One of these (150 kDa), produced in relatively small amounts, consists of a non-haem protein plus four haemoproteins (28, 33, 41 and 47 kDa). The second complex is a novel dimeric multi-haem cytochrome c (46 kDa) which constitutes about 25% of the periplasmic c-type cytochrome. It reacts with CO and has no methionine ligands. One subunit (16 kDa) has two low-spin haems; the larger subunit (30 kDa) has three haems which have low-spin characteristics in the oxidized state and are high-spin in the reduced state. The subunits were readily separated at pH 12 and could be subsequently reconstituted into a complex indistinguishable from the original. The 30 kDa subunit was denatured on prolonged exposure to high pH, which also converted it to a low-spin cytochrome. No function could be designated for these novel c-type cytochrome complexes.

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

Cited by 17 publications

References 29 publications

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

Isolation, sequencing, and mutagenesis of the gene encoding cytochrome c553i of Paracoccus denitrificans and characterization of the mutant strain

Characterization of the periplasmic cytochromes c of Paracoccus denitrificans: identification of the electron acceptor for methanol dehydrogenase, and description of a novel cytochrome c heterodimer

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