Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification.

  title={Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gln modification.},
  author={John S. Lloyd and Avninder S Bhambra and John Colin Murrell and Howard Dalton},
  journal={European journal of biochemistry},
  volume={248 1},
The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-length active form and truncated forms, B' and B". Electrospray ionisation mass spectrometry (ESI-MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N-terminus of protein B. This truncate was designated B' and molecular masses were assigned to proteins B and B' of 15,852.6+/-0.4 Da and 14,629.5+/-0.3 Da… 

Structure of the soluble methane monooxygenase regulatory protein B.

NMR spectroscopy is used to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system that catalyzes the selective oxidation of methane to methanol, providing structural insight into how the regulatory protein interacts with the hydroxylase.

Sequencing and analysis of the Methylococcus capsulatus (Bath) soluble methane monooxygenase genes

Alignments of homologous proteins from sMMO, phenol hydroxylase, toluene 2-, 3-, and 4-monooxygenases, and alkene monooxygenase systems revealed an interesting set of absolutely conserved amino-acid residues, including previously unidentified residues located outside the diiron active site of the hydoxylase.

Methane Monooxygenase Hydroxylase

Soluble methane monooxygenase (sMMO) catalyzes the conversion of methane and dioxygen to methanol and water. It is a cytoplasmic system of three proteins produced in methanotrophic bacteria grown

Molecular analysis of the soluble butane monooxygenase from 'Pseudomonas butanovora'.

'Pseudomonas butanovora' is capable of growth with butane via the oxidation of butane to 1-butanol, which is catalysed by a soluble butane monooxygenase (sBMO). In vitro oxidation of ethylene (an

Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

Substantial development of the method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO is reported, demonstrating proof of the principle for protein engineering of this uniquely versatile enzyme.

Molecular genetics and microbiology of bioremediation using methane-oxidising bacteria

Results from site-directed mutagenesis created four new mutants in addition to performing further characterization of another two, and indicated that radical chemistry at this position is not essential for monooxygenase activity toward a number of aliphatic and aromatic hydrocarbons.

Molecular biology and regulation of methane monooxygenase

A model for copper regulation based on results from Northern analysis, primer-extensions and new sequence data is presented, and a number of unanswered questions for future studies are raised.

Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.

This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains.

X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) are determined, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle.



Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath).

This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in the analysis of the assembly and catalysis of sMMO.

Resolution of the methane mono-oxygenase of Methylococcus capsulatus (Bath) into three components. Purification and properties of component C, a flavoprotein.

1. Ion-exchange chromatography resolves the methane mono-oxygenase from soluble extracts of Methylococcus capsulatus (Bath) into three fractions. 2. Fractions A and B are comparatively stable at 0

Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath)

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl

Purification and Properties of a Soluble Methane Monooxygenase from Methylocystis sp. M.

A soluble methane monooxygenase (sMMO: EC was purified from a type II obligate methanotroph, Methylocystis sp. M. Ion exchange chromatography elution separated the sMMO into three

Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph.

The data presented here provide the first evidence based on catalysis that the site of the monooxygenation reaction is located on the hydroxylase, and implies a new mechanism for the generation of highly reactive oxygen capable of insertion into unactivated carbon-hydrogen bonds.

Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b

The molecular characterization of the soluble MMO genes from the Type II methanotroph Methylosinus trichosporium OB3b, including the structural genes encoding the α, β and γ subunits of sMMO protein A and the structural gene encoding component B are detailed.