Purification and characterization of an inverting stereo- and enantioselective sec-alkylsulfatase from the gram-positive bacterium Rhodococcus ruber DSM 44541.
Whole cells of Rhodococcus ruber DSM 44541 were found to hydrolyze (+/-)-2-octyl sulfate in a stereo- and enantiospecific fashion. When growing on a complex medium, the cells produced two sec-alkylsulfatases and (at least) one prim-alkylsulfatase in the absence of an inducer, such as a sec-alkyl sulfate or a sec-alcohol. From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification. In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa. Maximal enzyme activity was observed at 30 degrees C and at pH 7.5. Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]). Enzymatic hydrolysis of (R)-2-octyl sulfate furnished (S)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom. Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear sec-alkyl sulfates (C(7) to C(10)) were freely accepted, no activity was found with branched and mixed aryl-alkyl sec-sulfates. Due to the fact that prim-sulfates were not accepted, the enzyme was classified as sec-alkylsulfatase (EC 3.1.6.X).
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria.
Mateja
M
Pogorevc
Kurt
K
Faber
D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D008970:Molecular Weight; D012240:Rhodococcus / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013429:Sulfatases / Q000302:isolation & purification* / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D013463:Sulfuric Acid Esters; D013429:Sulfatases; C019244:alkylsulfatase