A highly enantioselective and stereoselective secondary alkylsulfatase from Pseudomonas sp. DSM6611 (Pisa1) was heterologously expressed in Escherichia coli BL21, and purified to homogeneity for kinetic and structural studies. Structure determination of Pisa1 by X-ray crystallography showed that the protein belongs to the family of metallo-b-lactamases with a conserved binuclear Zn 2+ cluster in the active site. In contrast to a closely related alkylsulfatase from Pseudomonas aeruginosa (SdsA1), Pisa1 showed a preference for secondary rather than primary alkyl sulfates, and enantioselectively hydrolyzed the (R)-enantiomer of rac-2-octyl sulfate, yielding (S)-2-octanol with inversion of absolute configuration as a result of C-O bond cleavage. In order to elucidate the mechanism of inverting sulfate ester hydrolysis, for which no counterpart in chemical catalysis exists, we designed variants of Pisa1 guided by three-dimensional structure and docking experiments. In the course of these studies, we identified an invariant histidine (His317) near the sulfate-binding site as the general acid for crucial protonation of the sulfate leaving group. Additionally, amino acid replacements in the alkyl chain-binding pocket generated an enzyme variant that lost its stereoselectivity towards rac-2-octyl sulfate. These findings are discussed in light of the potential use of this enzyme family for applications in biocatalysis.
DatabaseThe atomic coordinates and structural factors have been deposited in the Protein Data Bank under the accession codes 2YHE (wild type, crystal form I), 4AV7 (double variant Ser233? Tyr/Ph250?Gly) and 4AXH (wild type, crystal form II)
Structured digital abstractPisa1 and Pisa1 bind by x-ray crystallography (View interaction)
The defect complexes that are formed when protons with energies in the MeV-range were implanted into highpurity silicon were investigated. After implantation, the samples were annealed at 400 °C or 450 °C for times ranging between 15 minutes and 30 hours. The resistivity of the samples was then analyzed by Spreading Resistance Profiling (SRP). The resistivity shows minima where there is a high carrier concentration and it is possible to extract the carrier concentration from the resistivity data. Initially, there is a large peak in the carrier concentration at the implantation depth where most of the hydrogen is concentrated. For longer anneals, this peak widens as the hydrogen diffuses away from the implantation depth. Following the changes in resistivity as a function of annealing time allows us to characterize the diffusion of hydrogen through these samples. Differences in the diffusion were observed depending on whether the silicon was grown by the magnetic Czochralski (m:Cz) method or the Float zone (Fz) method.
Protons with energies of 1 MeV and 2.5 MeV were implanted into a p-doped silicon wafer and then the wafer was annealed at 350 °C for one hour. This resulted in two n-doped layers in the otherwise p-doped sample. The carrier concentration was measured using spreading resistance profiling while the positions of the four pn-junctions were measured using electron beam induced current measurements. The carrier concentration is not limited by the available hydrogen but by the concentration of suitable radiation induced defects.
Electron beam induced current (EBIC) measurements were used to determine the doping type and to extract the diffusion length in proton implanted silicon wafers. This method makes it possible to distinguish between n-type and p-type at low carrier concentrations. Because of the defects caused by the implantation, the diffusion length is much smaller in the implanted than in the non-implanted regions.
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