Beschreibung:
<jats:sec><jats:label /><jats:p>A highly enantioselective and stereoselective secondary alkylsulfatase from <jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>seudomonas</jats:italic> sp. <jats:styled-content style="fixed-case">DSM</jats:styled-content>6611 (<jats:styled-content style="fixed-case">P</jats:styled-content>isa1) was heterologously expressed in <jats:italic><jats:styled-content style="fixed-case">E</jats:styled-content>scherichia coli</jats:italic> <jats:styled-content style="fixed-case">BL</jats:styled-content>21, and purified to homogeneity for kinetic and structural studies. Structure determination of <jats:styled-content style="fixed-case">P</jats:styled-content>isa1 by <jats:styled-content style="fixed-case">X</jats:styled-content>‐ray crystallography showed that the protein belongs to the family of metallo‐β‐lactamases with a conserved binuclear <jats:styled-content style="fixed-case">Z</jats:styled-content>n<jats:sup>2+</jats:sup> cluster in the active site. In contrast to a closely related alkylsulfatase from <jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>seudomonas aeruginosa</jats:italic> (<jats:styled-content style="fixed-case">S</jats:styled-content>ds<jats:styled-content style="fixed-case">A</jats:styled-content>1), <jats:styled-content style="fixed-case">P</jats:styled-content>isa1 showed a preference for secondary rather than primary alkyl sulfates, and enantioselectively hydrolyzed the (<jats:italic><jats:styled-content style="fixed-case">R</jats:styled-content></jats:italic>)‐enantiomer of <jats:italic>rac</jats:italic>‐2‐octyl sulfate, yielding (<jats:italic><jats:styled-content style="fixed-case">S</jats:styled-content></jats:italic>)‐2‐octanol with inversion of absolute configuration as a result of <jats:styled-content style="fixed-case">C</jats:styled-content>–<jats:styled-content style="fixed-case">O</jats:styled-content> 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 <jats:styled-content style="fixed-case">P</jats:styled-content>isa1 guided by three‐dimensional structure and docking experiments. In the course of these studies, we identified an invariant histidine (<jats:styled-content style="fixed-case">H</jats:styled-content>is317) 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 <jats:italic>rac</jats:italic>‐2‐octyl sulfate. These findings are discussed in light of the potential use of this enzyme family for applications in biocatalysis.</jats:p></jats:sec><jats:sec><jats:title>Database</jats:title><jats:p>The atomic coordinates and structural factors have been deposited in the <jats:styled-content style="fixed-case">P</jats:styled-content>rotein <jats:styled-content style="fixed-case">D</jats:styled-content>ata <jats:styled-content style="fixed-case">B</jats:styled-content>ank under the accession codes <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2YHE">2YHE</jats:ext-link> (wild type, crystal form I), <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4AV7">4AV7</jats:ext-link> (double variant <jats:styled-content style="fixed-case">S</jats:styled-content>er233→<jats:styled-content style="fixed-case">T</jats:styled-content>yr/<jats:styled-content style="fixed-case">P</jats:styled-content>h250→<jats:styled-content style="fixed-case">G</jats:styled-content>ly) and <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4AXH">4AXH</jats:ext-link> (wild type, crystal form II)</jats:p></jats:sec><jats:sec><jats:title>Structured digital abstract</jats:title><jats:p>
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<jats:list-item><jats:p><jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.uniprot.org/uniprot/F8KAY7">Pisa1</jats:ext-link> and <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.uniprot.org/uniprot/F8KAY7">Pisa1</jats:ext-link> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407">bind</jats:ext-link> by <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114">x-ray crystallography</jats:ext-link> (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8397985">View interaction</jats:ext-link>)</jats:p></jats:list-item>
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