Dimethylsulfoniopropionate-dependent demethylase (DmdA) from Pelagibacter ubique and Silicibacter pomeroyi.
The ubiquitous algal metabolite dimethylsulfoniopropionate (DMSP) is a major source of carbon and reduced sulfur for marine bacteria. Recently, the enzyme responsible for the demethylation of DMSP, designated DmdA, was identified, and homologs were found to be common in marine bacterioplankton cells. The recombinant DmdA proteins from the cultured marine bacteria Pelagibacter ubique HTCC1062 and Silicibacter pomeroyi DSS-3 were purified with a three-step procedure using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatographies. The P. ubique enzyme possessed an M(r) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 38,500. Under nondenaturing conditions, the M(r) was 68,000, suggesting that the enzyme was likely to be a dimer. The purified enzyme exhibited strict substrate specificity for DMSP, as DmdA from both S. pomeroyi and P. ubique possessed no detectable demethylase activity with glycine betaine, dimethyl glycine, methylmercaptopropionate, methionine, or dimethylsulfonioacetate. Less than 1% activity was found with dimethylsulfoniobutanoate and dimethylsulfoniopentanoate. The apparent K(m)s for DMSP were 13.2 +/- 2.0 and 5.4 +/- 2.3 mM for the P. ubique and S. pomeroyi enzymes, respectively. In cell extracts of S. pomeroyi DSS-3, the apparent K(m) for DMSP was 8.6 +/- 1.2 mM, similar to that of purified recombinant DmdA. The intracellular concentration of DMSP in chemostat-grown S. pomeroyi DSS-3 was 70 mM. These results suggest that marine bacterioplankton may actively accumulate DMSP to osmotically significant concentrations that favor near-maximal rates of DMSP demethylation activity.
Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
Chris R
CR
Reisch
Mary Ann
MA
Moran
William B
WB
Whitman
D020561:Alphaproteobacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D041902:Rhodobacteraceae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013452:Sulfonium Compounds / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013452:Sulfonium Compounds; C068078:dimethylpropiothetin; D010088:Oxidoreductases