Structure and mechanism of an inverting alkylsulfatase from Pseudomonas sp. DSM6611 specific for secondary alkyl sulfates

Abstract

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-β-lactamases with a conserved binuclear Zn2+ 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. Database The 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 abstract Pisa1 and Pisa1 bind by x-ray crystallography (View interaction) The structure of a highly enantioselective and stereoselective secondary alkylsulfatase, which hydrolyses 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, was solved. An invariant histidine residue near the sulfate binding site was identified as the general acid for crucial protonation of the sulfate leaving group © 2012 The Authors Journal compilation © 2012 FEBS.