Stereo-Complementary sec Alkyl Sulfatases

Depending on the type of enzyme and its underlying catalytic mechanism, the enzymatic hydrolysis of sec-alkyl sulfate esters catalyzed by sulfatases may proceed through inversion or retention of configuration via scission of the C-O- versus the S-O-bond. This stereo-complementarity makes them ideal catalysts for the deracemization of sec- alcohols, during which a racemate is quantitatively converted into a single stereoisomerically pure product without the occurrence of an `unwanted` stereoisomer. Starting from our first successful observations on the stereo- complementarity of the biohydrolysis of sec-alkyl sulfate esters (`proof of principle`), the full potential of microbial sulfatases for preparative-scale organic chemistry will be evaluated. Guided by genome analyses, the substrate- spectrum, stereo-preference and stereochemistry of sulfatases from the following biological sources will be investigated: (1) Inverting sulfatases from bacteria (especially Actinomycetes) and sulfur-metabolizing extremophilic Archaea. (2) Retaining sulfatases from marine Planctomycetes and Basidiomycetes. The biochemical characterization of selected sulfatases will provide important contributions to the elucidation of (hitherto unknown) catalytic mechanism of inverting sulfatases. Stereo-complementary sulfatases (acting with inversion versus retention) will be employed for the development of deracemization processes for sec-alcohols: thus, whereas one enantiomer from the racemate is hydrolyzed with inversion of configuration, the remaining non-reacting mirror-image counterpart is converted with retention to yield a single stereoisomeric product in quantitative yield. The applicability of this technique will be demonstrated for selected sec-alcohols on a preparative scale. Overall, this project provides important contributions to the enzymology of sulfatases and the development of enviromentally benign (`green`) processes for synthetic organic chemistry.

During the course of the project we were able to isolate and overexpress the first member of a scarcely investigated enzyme family: the alkylsulfatases. These enzymes catalyse the hydrolysis of alkylsulfate esters, which are widely used in daily life as household detergents and as shampoo ingredients. Alkyl sulfatases are unique in one respect, as they are able to either conserve (`retain`) or flip over (`invert`) the three-dimensional structure of their substrates during catalysis, which may be compared with the flipping of an umbrella by a gust of wind. In contrast, the majority of sulfatases studied to date conserve (`retain`) the substrate arrangement. The protein was detected in a Pseudomonas bacterium and was isolated by protein purification via several steps. Using the amino acid and genomic sequence, this protein could be cloned into a simple Escherichia coli host, which allowed the production of sufficient amounts for characterisation. The mode of catalysis of the protein termed `PISA1` (Pseudomonas inverting sec-alkylsulfatase 1) was closely investigated by using water bearing a `heavy` ( 18O) oxygen. Mass analysis of the products revealed that the oxygen was indeed incorporated into the carbon framework (and not into sulfate), which proved that the spatial arrangement of the substrate (the `absolute configuration`) was flipped (`inverted`) during catalysis. It is remarkable that this reaction cannot be performed by using traditional (chemical) methods of catalysis. PISA1 can be employed to convert a mirror-image mixture of a secondary alkylsulfate ester, e.g. derived by conventional chemical synthesis, into a single pure isomer of an alcohol without occurrence of an unwanted by- product, which provides an elegant - `waste-free` - method of chemical production.