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-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)

Crystal structure of thermostable alkylsulfatase SdsAP from Pseudomonas sp. S9

“…As reported in Pisa1, Ser 233 and Phe 250 are important residues to its preference for shorter alkyl chains, leading Pisa1 as a secondary alkylsulfatase [17]. However, in the equivalent position, there are Tyr 246 and Gly 263 in SdsAP, while Tyr 223 and Gly 240 in SdsA1 (Figure 5B).…”

“…Till now, the three-dimensional structures of type III alkylsulfatases, SdsA1 and Pisa1, have been characterized and interpreted [9,17]. They show a high structural similarity and a slight difference in their active site region.…”

“…As described, they share a distinct metallo-β-lactamase fold domain, a dimerization domain and a sterol carrier protein type 2 (SCP-2)-like fold domain. It has been suggested that SdsA1 is a primary alkylsulfatase preferring the hydrolysis of ‘primary alkyl’, while Pisa1 is a secondary alkylsulfatase preferring the hydrolysis of ‘secondary alkyl’ sulfates[9,17]. Recently, a novel sulfatase, SdsAP, was identified from a newly isolated bacterium Pseudomonas sp.…”

Structural and functional analysis show that the Escherichia coli uncharacterized protein YjcS is likely an alkylsulfatase

“…8,14 The sequence alignment exhibits a conserved HXHXDH catalytic motif among these proteins (Fig. 3).…”

“…SdsA1 is a primary alkylsulfatase, which enables the bacterium to degrade primary alkylsulfate SDS, whereas Pisa1 is an inverting alkylsulfatase specific for secondary alkylsulfates. 8,14 Recently, research attention has focussed on an effective bioremediation of detergents, especially SDS, because it is widely used in industrial settings and daily life, and is detrimental to the environment. [15][16][17][18] The pathway of SDS degradation is well documented in which SDS degradation is initiated with alkylsulfatase and the product, 1-dodecanol (1DO), is subsequently oxidized to 1-dodecanoic acid.…”