Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Cross AR

PROVIDER: S-EPMC1162356 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:The cytochrome complement of Methylophilus methylotrophus and its respiratory properties were determined during batch culture and in continuous culture under conditions of methanol-, nitrogen- and O(2)-limitation. About 35% of the cytochrome c produced by the bacteria was released into the growth medium, and of the remaining cytochrome c about half was membrane-bound and half was soluble. Two cytochromes c were always present on membranes (redox potentials 375mV and 310mV), and these probably correspond to the soluble cytochromes c described previously [Cross & Anthony (1980) Biochem. J.192, 421-427]. A third minor component of cytochrome c (midpoint potential 356mV) was only detected on membranes of methanol-limited bacteria. M. methylotrophus always contained two membrane-bound cytochromes b with alpha-band absorption maxima of about 556 and 563nm (measured at room temperature) and midpoint potentials of 110 and 60mV respectively. There appeared to be relatively more of the cytochrome b(563) in methanol-limited bacteria. A third b-type cytochrome with an alpha-band absorption maximum at 558 (at 77K) reacted with CO and had a high midpoint redox potential (260mV); it is thus a potential oxidase and hence is called cytochrome o. The roles of these cytochromes in electron transport were confirmed by investigating the patterns of respiratory inhibition. It is proposed that two cytochromes are physiological oxidases: cytochrome a+a(3), which is present only in methanol-limited conditions, and the cytochrome o, which is induced 10-fold in conditions of methanol excess. Schemes for electron transport from methanol and NAD(P)H to O(2) in M. methylotrophus under various limitations are proposed. Spectra and potentiometric titrations of cytochromes in whole cells and membranes of M. methylotrophus grown under various nutrient limitations have been deposited as Supplementary Publication SUP 50111 (10 pages) 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. (1978) 169, 5.

Project description:MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5'-TCCRAC-3' (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N(6)-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D(70)-X(9)-EXK(82), characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N(6)-adenine gamma-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets