Dataset Information

Structure of the redox sensor domain of Methylococcus capsulatus (Bath) MmoS.

ABSTRACT: MmoS from Methylococcus capsulatus (Bath) is the multidomain sensor protein of a two-component signaling system proposed to play a role in the copper-mediated regulation of soluble methane monooxygenase (sMMO). MmoS binds an FAD cofactor within its N-terminal tandem Per-Arnt-Sim (PAS) domains, suggesting that it functions as a redox sensor. The crystal structure of the MmoS tandem PAS domains, designated PAS-A and PAS-B, has been determined to 2.34 A resolution. Both domains adopt the typical PAS domain alpha/beta topology and are structurally similar. The two domains are linked by a long alpha helix and do not interact with one another. The FAD cofactor is housed solely within PAS-A and is stabilized by an extended hydrogen bonding network. The overall fold of PAS-A is similar to those of other flavin-containing PAS domains, but homodimeric interactions in other structures are not observed in the MmoS sensor, which crystallized as a monomer. The structure both provides new insight into the architecture of tandem PAS domains and suggests specific residues that may play a role in MmoS FAD redox chemistry and subsequent signal transduction.

SUBMITTER: Ukaegbu UE

PROVIDER: S-EPMC2707821 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Structure of the redox sensor domain of Methylococcus capsulatus (Bath) MmoS.

Ukaegbu Uchechi E UE Rosenzweig Amy C AC

Biochemistry 20090301 10

MmoS from Methylococcus capsulatus (Bath) is the multidomain sensor protein of a two-component signaling system proposed to play a role in the copper-mediated regulation of soluble methane monooxygenase (sMMO). MmoS binds an FAD cofactor within its N-terminal tandem Per-Arnt-Sim (PAS) domains, suggesting that it functions as a redox sensor. The crystal structure of the MmoS tandem PAS domains, designated PAS-A and PAS-B, has been determined to 2.34 A resolution. Both domains adopt the typical PA ...[more]

PMID: 19271777

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Project description:P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231-244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879-5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12, 000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457-460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.

Project description:Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the ?-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath).IMPORTANCE Methanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such as Escherichia coli Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection in M. capsulatus (Bath) by employing pheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants of M. capsulatus (Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.

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Dataset Information

Structure of the redox sensor domain of Methylococcus capsulatus (Bath) MmoS.

Publications

Structure of the redox sensor domain of Methylococcus capsulatus (Bath) MmoS.

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