Transformations of Dimethylsulfide - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 298

2.4 Dimethylsulfide Monooxygenases

2.4.1 Properties of Dimethylsulfide Monooxygenases

Hyphomicrobium species were amongst the earliest characterized DMS-degrading
bacteria to be discovered. Several strains of DMS-degrading Hyphomicrobium
species have been characterized physiologically (e.g., [ 16 , 33 , 37 , 38 , 183 ]).
Some of these may also degrade other methylated sulfur compounds including
dimethylsulfone and dimethylsulfoxide, which are initially reduced to DMS before
a DMS monooxygenase (DMSMO) catalyzes its degradation to MT and formalde-
hyde [ 16 , 38 ]. The activity of DMS monooxygenase was first identified in
Hyphomicrobium S[ 16 ] based on NADH-dependent oxygen uptake in the presence
of DMS, but the enzyme had not been purified and characterized until recently
[ 184 ], and, similar to the situation with DMS dehydrogenase, there is currently only
one characterized DMS monooxygenase.
The DMS monooxygenase from Hyphomicrobium sulfonivorans S1 was shown
to be a flavin-dependent monooxygenase composed of two subunits (DmoAB) with
molecular masses of 53 and 19 kDa, respectively. The large subunit DmoA is an
FMN-dependent monooxygenase, while the small subunit is a NAD(P)H-dependent
flavin oxidoreductase. Addition of FMN to the enzyme assay increased the enzyme
activity approximately 12-fold. Activity assays with different combinations of
reduced nicotinamides and flavin cofactors suggested that the small subunit is an
NADH-dependent FMN reductase, although some activity was also shown with
NADPH as electron donor. Use of alternative flavin cofactors resulted in negligible
enzyme activities in line with the prediction that FMN is the flavin cofactor [ 184 ].
DMSMO converted DMS to MT and formaldehyde and had decreasing activity
when alternative alkylsulfides with increasing length of the alkyl chain were used as
substrates. Chelation experiments with EDTA, EGTA, and bathocuproine all
decreased the enzyme activity. Reconstitution with a range of divalent metal
cations showed the best restoration of enzyme activity (63 % compared to the
native enzyme) when Fe 2+ and Mg 2+ were added in combination suggesting both
are required for the activity of the enzyme, but further characterization of the metal
content of DMSMO is still required [ 184 ].
DMS monooxygenase had a K M of approximately 17

ʼ M for DMS and a V max of

mol min 1 mg 1 protein giving a k cat of 5.45 s 1 . This relatively low turnover
number of DMSMO appears to be compensated for by a relatively high abundance
of the enzyme which was estimated to be nearly 9 % of cell protein [ 184 ].
The large subunit was further characterized by peptide sequencing and gene
cloning. PCR primers were designed based on peptide sequences and allowed
identification of cloned genomic DNA fragments containing the gene encoding
the DMSMO large subunit ( dmoA ). The genetic context of dmoA was shown to
contain two genes encoding predicted FMN reductases, which may encode the
small subunit of the enzyme (DmoB), but the dmoB gene has not been identified
unequivocally to date due to lack of definitive mass spectrometry data and failure of

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