Biochemical characterisation of methanethiol oxidase: a key enzyme of volatile organosulfur compound degradation

Lead Research Organisation: University of Warwick
Department Name: Warwick HRI

Abstract

We propose to characterise the properties of a bacterial enzyme that converts a foul smelling, toxic gaseous organic sulfur compound. Methanethiol oxidase converts methanethiol - a gas smelling of rotten cabbage - which is an important sulfur compound in nature. The products of the methanethiol catalysed reaction are formaldehyde, hydrogen sulfide (smells of rotten eggs) and hydrogen peroxide (a chemical that is used as a bleaching agent in contact lens disinfection fluids). Very little is known of the molecular and catalytic properties of methanethiol oxidases. The work we propose will provide fundamental insights into the biochemistry of methanethiol degradation, a process that on a global scale is responsible for turnover of some 300-500 million tons of sulfur in the oceans alone, where methanethiol occurs as a degradation product of a climate cooling gas (dimethylsulfide) and its precursor molecule (dimethylsulfoniopropionate). Methanethiol plays a role as a flavour compound in foods and beverages, but can also contribute to what we perceive as off-flavours when it occurs at too high concentrations. In an industrial context, methanethiol is also significant as a degradation product of bulk solvents (dimethylsulfoxide) and it is conceivable that methanethiol oxidase could find application as an industrial biocatalyst. Our preliminary data also indicate that this enzyme may work in concert with other enzymes in a complex of proteins inside the cell, which may protect it to some degree from its toxic intermediates and may enhance its function. This enzyme complex will also be studied further. An interesting aspect of methanethiol oxidase that we have just discovered is that this bacterial enzyme is encoded by a gene for which there has not been any information indicating its function previously. It is hence of interest to further investigate the enzyme, in particular as there are similar enzymes encoded by genomes of animals including humans on whose function similarly little is known. Thus, studying this bacterial enzyme in more detail, may shed light on the evolution of a type of protein which is also found in our bodies. The ultimate goal of characterising an enzyme is to elucidate its three dimensional structure. While this is not planned in this project, we aim to make the first step towards achieving that goal by producing crystals of the enzyme that can be characterised by X-ray diffraction analysis. This will require using recombinant DNA technology, which will allow to produce sufficient amounts of the enzyme for crystallisation.

Technical Summary

The enzyme methanethiol oxidase (MTO) converts methanethiol to formaldehyde, hydrogen sulfide and hydrogen peroxide. MTO is a key enzyme in the biogeochemical cycle of sulfur converting organically bound sulfur to inorganic sulfur. We have purified MTO from Hyphomicrobium species VS and our preliminary analysis has provided important new insights that challenge previous studies of homologous enzymes which are summarised below. Our data show that: - MTO is composed of four identical 46 kDa subunits rather than a monomer as previously suggested - is not devoid of metals, as each enzyme molecule contains 4 atoms of Calcium and 2 atoms of Copper We have also identified the gene encoding the enzyme for the first time. Analysis of the gene sequence shows MTO to be a member of the poorly characterised family of so-called 'Selenium-binding proteins' for which no function has been identified in prokaryotes and which are thought to be involved in intra golgi transport in eukaryotes. Prediction of the fold of the protein further suggest this to be a beta propeller with similarities to methanol dehydrogenase and methylamine dehydrogenase which also catalyse the degradation of reduced one-carbon compounds. Previously, MTO was thought not to require a cofactor. Our preliminary UV-vis spectrophotometric and fluorescence spectroscopy analysis indicate the presence of a associated chromophores which could indicate the presence of a PQQ-like cofactor. We propose to built on these data and characterise in detail the biochemical properties of MTO, assess the role of the metals for activity of the enzyme, carry out detailed spectrohotometric and fluorescence spectroscopy studies which will provide new insights into the biochemistry of the enzyme. We will further assess the association of MTO with other proteins in an enzyme complex and establish a heterologous expression system with a view of producing amounts of MTO suitable for crystallisation.

Publications

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Description Methanethiol (MT) is an intermediate of the bacterial metabolism of both dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS) and its degradation in bacteria is catalyzed by methanethiol oxidase (MTO) producing hydrogen sulfide, hydrogen peroxide and formaldehyde. Bacterial MTOs have remained poorly characterised. In this project we identifed and characterised the genetic and biochemical basis of bacterial MT oxidase through characterization of MTO from a DMS degrader (Hyphomicrobium VS). We have established the subcellular localisation, the oligomeric structure, kinetic parameters, secondary structural motifs, metal content, and genetic basis of MTO of Hyphomicrobium VS.

Based on our findings on the MTO enzyme and its encoding gene, we also investigated the function of MTO in a marine bacterium (Ruegeria pomeroyi DSS3) in collaboration with the group of Johnston and Todd at University of East Anglia. Genetic characterisation included the knock out of the gene which resulted in a MTO defective mutant. Transcriptional regulation of the gene was also assessed.
Exploitation Route This enzyme may find use in biofiltration of odorous airstreams contaminated with MT; it may also be useful as a biosensor This enzyme may find use in biofiltration of odorous airstreams contaminated with MT; it may also be useful as a biosensor; identification of the gene allows the analysis of the identity and distribution of MT oxidising bacteria in the environment. Based on the identification and characterisation of the MTO in bacteria, collaborators at the Radboud University in Nijmegen (NL) identified a homologue of the MTO gene in humans. The human MTO is encoded by SELENBP1 and mutations in this gene have been identified as a cause for hereditary extraoral halitosis.
Sectors Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description DIAMOND Beamtime
Amount £57,564 (GBP)
Funding ID SP8769-1 
Organisation Diamond Light Source 
Sector Academic/University
Country United Kingdom
Start 09/2013 
End 09/2013
 
Description Human SELENBP1 
Organisation Radboud University Nijmegen Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution Partners at Radbound are Professor Ron Wevers, Dr Huub op den Camp, Dr Arjan Pol. Our contribution to their research was helping to identify the gene encoding the human methanethiol oxidase, which was the basis of further research by the Radboud team which identified mutations as in that gene as a cause in a hereditary form of extraoral halitosis.
Collaborator Contribution Detailed study of the phenotype of patients presenting with extraoral halitosis (bad breath, not caused by poor oral hygiene).
Impact Pol et al. 2018. Nature Genetics. See publications section for full details.
Start Year 2011