Identification of the gene encoding the regulatory protein B of soluble methane monooxygenase

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions. The maximum catalytic activity of hydroxylase (MMOH) is achieved through the interplay of its regulatory protein (MMOB) and reductase. An additional auxiliary protein, MMOD, functions as an inhibitor of MMOH; however, its inhibitory mechanism remains unknown. Here, we report the crystal structure of the MMOH-MMOD complex from Methylosinus sporium strain 5 (2.6 Å). Its structure illustrates that MMOD associates with the canyon region of MMOH where MMOB binds. Although MMOD and MMOB recognize the same binding site, each binding component triggers different conformational changes toward MMOH, which then respectively lead to the inhibition and activation of MMOH. Particularly, MMOD binding perturbs the di-iron geometry by inducing two major MMOH conformational changes, i.e., MMOH β subunit disorganization and subsequent His147 dissociation with Fe1 coordination. Furthermore, 1,6-hexanediol, a mimic of the products of sMMO, reveals the substrate access route.

Download Full-text

A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

Applied and Environmental Microbiology ◽

10.1128/aem.03884-15 ◽

2016 ◽

Vol 82 (6) ◽

pp. 1917-1923 ◽

Cited By ~ 25

Author(s):

Wenyu Gu ◽

Muhammad Farhan Ul Haque ◽

Bipin S. Baral ◽

Erick A. Turpin ◽

Nathan L. Bandow ◽

...

Keyword(s):

Methane Monooxygenase ◽

Copper Uptake ◽

Wild Type ◽

Particulate Methane Monooxygenase ◽

Methylosinus Trichosporium Ob3b ◽

Gene Encoding ◽

Methylosinus Trichosporium ◽

Content Type ◽

Soluble Methane Monooxygenase ◽

Genes Encoding

ABSTRACTMethanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin,mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well asmbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine ifmbnTis truly responsible for methanobactin uptake, a knockout was constructed inMethylosinus trichosporiumOB3b using marker exchange mutagenesis. The resultingM. trichosporiummbnT::Gmrmutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression ofmmoXandpmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-typeM. trichosporiumOB3b, indicating that the TonB-dependent transporter encoded bymbnTis responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When thembnT::Gmrmutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-typeM. trichosporiumOB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.

Download Full-text

rpoN, mmoR and mmoG, genes involved in regulating the expression of soluble methane monooxygenase in Methylosinus trichosporium OB3b

Microbiology ◽

10.1099/mic.0.26060-0 ◽

2003 ◽

Vol 149 (7) ◽

pp. 1771-1784 ◽

Cited By ~ 48

Author(s):

Graham P. Stafford ◽

Julie Scanlan ◽

Ian R. McDonald ◽

J. Colin Murrell

Keyword(s):

Methane Monooxygenase ◽

Regulatory Gene ◽

Soluble Form ◽

Gene Encoding ◽

Independent Manner ◽

Methylosinus Trichosporium ◽

Marker Exchange ◽

Soluble Methane Monooxygenase ◽

Genes Encoding

The methanotrophic bacterium Methylosinus trichosporium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at low copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a σ N promoter 5′ of mmoX. In this study the genes encoding σ N (rpoN) and a typical σ N-dependent transcriptional activator (mmoR) were cloned and sequenced. mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5′ of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that σ N plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a σ N- and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.

Download Full-text

MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase

10.1101/331512 ◽

2018 ◽

Cited By ~ 2

Author(s):

Hanseong Kim ◽

Sojin An ◽

Yeo Reum Park ◽

Hara Jang ◽

Sang Ho Park ◽

...

Keyword(s):

Crystal Structure ◽

Catalytic Activity ◽

Structural Changes ◽

Methane Monooxygenase ◽

Regulatory Protein ◽

Inhibitory Mechanism ◽

Ambient Conditions ◽

Soluble Methane Monooxygenase ◽

Access Channel ◽

Hydrocarbon Substrates

SummarySoluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions1-3. The maximum catalytic activity of hydroxylase (MMOH) is achieved via interplay of its regulatory protein (MMOB) and reductase4-6. An additional auxiliary protein, MMOD, is believed to function as an inhibitor of the catalytic activity of MMOH; however, the mechanism of its action remains unknown7,8. Herein, we report the crystal structure of MMOH–MMOD complex fromMethylosinus sporiumstrain 5 (2.6 Å), which illustrates that two molecules of MMOD associate symmetrically with the canyon region of MMOH in a manner similar to MMOB, indicating that MMOD competes with MMOB for MMOH recognition. Further, MMOD binding disrupts the geometry of the di-iron centre and opens the substrate access channel. Notably, the electron density of 1,6-hexanediol at the substrate access channel mimics products of sMMO in hydrocarbon oxidation. The crystal structure of MMOH–MMOD unravels the inhibitory mechanism by which MMOD suppresses the MMOH catalytic activity, and reveals how hydrocarbon substrates/products access to the di-iron centre.

Download Full-text