Expression and characterisation of methanethiol oxidase and ...

mexicorubberBiotechnology

Feb 20, 2013 (4 years and 1 month ago)

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Principal Supervisor name and department
:

Hendrik Schäfer, SLS


Second Supervisor name and department:

Vilmos Fulop, SLS

Tim Bugg, Chemistry


Where will
the student be based
?

School of Life Sciences, Warwick


PhD project title:

Expression and
characterisation of
methanethiol oxidase and evaluation of its structure
-
function
relationship and biotechnological potential


Project description:

Methanethiol (MT), a volatile organic sulphur compound, represents a key compound in the biogeochemical cycl
e
of sulphur, for instance as a metabolic intermediate of microbial metabolism of dimethylsulfoniopropionate
(DMSP) and dimethylsulfide (DMS), but also of industrial bulk chemicals such as dimethylsulfoxide (DMSO).
Methanethiol is further of relevance as a

degradation intermediate of DMSO, a
n important industrial solvent, and
as a compound that can impart desirable aromas to certain food stuffs or cause off
-
flavours at too high
concentrations for instance in brewing and wine production as well as in the wat
er industry.


D
egradation
of MT
in bacteria is catalyzed by methanethiol oxidase (MTO) producing hydrogen sulfide, hydrogen
peroxide

and formaldehyde.
The biotechnological potential of this enzyme has yet to be fully evaluated, but there
is considerable potential of using the enzyme as a specific biosensor for methanethiol, or in engineering of
bacterial strains capable of improved biofiltration of volat
ile organic sulphur compound emissions. The
biochemical and genetic basis of methanethiol oxidation has remained poorly characterised (Bentley and
Chasteen, 2004; Schäfer
et al.
, 2010). Recently, HS and TB (with collaborators at Radboud University Nijmegen
,
NL) have made significant progress in understanding of MTO biochemistry. MTO of
Hyphomicrobium
VS
(Pol et
al 1004)
is a soluble enzyme
. T
he presence of an N
-
terminal signal peptide and experimentally determined N
-
terminal sequence
suggests it is

periplas
m
ic
.
A
ctive MTO preparation contains Ca and Cu and, with collaborator
Andersson (Oslo, Norway), we have shown using electron paramagnetic resonance spectroscopy that Cu
undergoes reduction/oxidation during the catalytic cycle. We have identified the gene e
ncoding MTO (
mtoX
) and
have assembled a draft genome sequence of
H
. VS. Comparative genomics analyses revealed the existence of a
conserved
mto
locus in a number of bacteria that have the ability to degrade MT. The putative
mto
operon has
genes encoding a
SCO1/SenC domain protein and a MauG protein downstream of
mtoX
, the products of
which

are
periplasmic
too
. SCO1
likely
encodes a copper chaperone deliver
ing

Cu atoms required in MTO
whereas

M
auG
, a diheme cytochrome
c
peroxidase homologue,
is likely

involv
ed in posttranslational modification giv
ing

rise to a protein derived tryptophan tryptophylquinone (TTQ) cofactor, akin to that of methylamine dehydrogenase
(Davidson, 2007).
While there is no

recognised Cu binding site in MTO
,

the
mto
-
associated SCO1 ha
s
a
modification of the conserved

Cu binding motifs
, with

CXXXXXC rather than the canonical CXXXC
.


Further work is required to advance our understanding of the biochemistry of MTO oxidation.
The aim of

this
project

is to
express
MTO and associated
maturation proteins
, affinity purify the recombinant proteins and
produce functional MTO through
in vitro
modification of the preMTO using SCO1 and MauG enzymes.
This will
allow us to investigate structure
-
function relationships

of MTO (crystallization, si
te directed mutagenesis)
, copper
binding by SCO1 and MTO,

and the biotechnological potential of the recombinant enzyme
.



Key
experimental skill
s

involved
:


Molecular cloning

Expression of proteins

Protein purification and character
isation

Gas
chromatography

Crystallisation, structural analysis, structural modelling

Site directed mutagenesis


References:


Bentley R, Chasteen TG (2004).
Environmental VOSCs
-

formation and degradation of dimethyl sulfide,
methanethiol and related materials.
Chemosphere
55:
291
-
317.

Davidson V (2007).
Protein
-
derived cofactors. Expanding the scope of post
-
translational modifications.
Biochemistry
46: 5283
-
5292.

Pol A, Op den Camp HJM, Mees SGM, Kersten MASH, van der Drift C (1994).
Isolation of a
dimethylsulfide
-
utilizing
Hyphomicrobium

species and its application in biofiltration of polluted air.
Biodegradation
5:
105
-
112.

Schäfer H, Myronova N, Boden R (2010).
Microbial degradation of dimethylsulphide and related C1
-
sulphur
compounds: organisms a
nd pathways controlling fluxes of sulphur in the biosphere.
Journal of Experimental

Botany
61:
315
-
334.



Contact details for application enquiries:

H.Schaefer@warwick.ac.uk

Telephone: 02476 575052



Keywords:

Sulphur biotechnology, methanethiol, methylmercaptan,
structure
-
function, structural biology,
biocatalyst, microbiology, molecular microbiology, biochemistry



Please state below which hazards may be connected with this studentship:



The
studentship will entail work involving:




if applies

Chemicals


high toxicity and category 1 or 2 substances



Organo
-
phosphate or carbamate pesticides



Skin or respiratory sensitising agents (e.g. insect parts, organic
dusts from animals, spores, pollen, antibiotics, fibres, chemical
sensitisers, wood dust etc)



Radionuclides


Significant manual handling


Mechanical repetition where the frequency and
duration are
significant


Working in areas where there are temperature extremes


Driving vehicles (tractors, fork lifts, ATVs etc)


Crop planting, harvesting, recording or grading


Working in close proximity to bees or other stinging insects


Working at height [>2 m] (using various types of access
equipment)


Working with noisy or vibrating equipment


Working at night (between 11.00pm and 6.00am)


Food handling



Other significant hazards (specify)