Purification and Characterization of an Inverting Stereo- and Enantioselective sec -Alkylsulfatase from the Gram-Positive Bacterium Rhodococcus ruber DSM 44541

Abstract

Whole cells of Rhodococcus ruber DSM 44541 were found to hydrolyze (؎)-2-octyl sulfate in a stereo-and enantiospecific fashion. When growing on a complex medium, the cells produced two sec-alkylsulfatases and (at least) one prim-alkylsulfatase in the absence of an inducer, such as a sec-alkyl sulfate or a sec-alcohol. From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification. In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa. Maximal enzyme activity was observed at 30°C and at pH 7.5. Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]). Enzymatic hydrolysis of (R)-2-octyl sulfate furnished (S)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom. Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear sec-alkyl sulfates (C 7 to C 10 ) were freely accepted, no activity was found with branched and mixed aryl-alkyl sec-sulfates. Due to the fact that prim-sulfates were not accepted, the enzyme was classified as sec-alkylsulfatase (EC 3.1.6.X).Sulfatases (EC 3.1.6.X) catalyze hydrolytic cleavage of the sulfate ester bond by liberating inorganic sulfate and the corresponding alcohol (Fig. 1). They are present in a wide variety of species, ranging from bacteria to humans (18,25). In contrast to the role of human sulfatases, which are involved in the desulfation of sulfated glycolipids, glycosaminoglycans, and steroids (7,19), the primary role of bacterial sulfatases is in the assimilation of sulfur (9, 12) or in the provision of carbon and/or energy sources for cell growth (15, 16).Our knowledge concerning bacterial sulfatases was summarized in an extensive review in 1982 (12). Alkylsulfatases hydrolyze organic sulfate esters of primary or secondary alkyl alcohols. Most of the work on alkylsulfatases was carried out with two gram-negative bacteria, Pseudomonas sp. strain C12B (ϭ NCIMB 11753 ϭ ATCC 43648) and Comamonas terrigena NCIMB 8193. It was shown that under appropriate culture conditions Pseudomonas sp. strain C12B produced up to five different alkylsulfatases (three secondary and two primary alkylsulfatases, which are specific for the hydrolysis of sec-and prim-alkyl sulfates, respectively) (2, 6, 11). In contrast, C. terrigena produced only two secondary alkylsulfatases irrespective of the culture conditions employed (1, 14). Some of these enzymes have been purified to homogeneity (2, 21).Alkylsulfatases have mostly been fou...

Contribution of Horizontal Gene Transfer to the Evolution of Saccharomyces cerevisiae

“…Though previously of unknown function, BDS1 has high sequence identity at the protein level to bacterial alkyl-sulfatases. Sulfatases catalyze hydrolytic cleavage of sulfate ester bonds, liberating sulfate and the corresponding alcohol (41). They are present in a wide variety of species, ranging from bacteria to humans.…”

“…In N. crassa aryl-sulfatase is up regulated by sulfur starvation and appears to function as a mechanism for sulfur scavenging. The primary roles of bacterial sulfatases are in assimilation of sulfur and in the provision of carbon (23,41).…”

Sulfatasen: Struktur, Mechanismus, biologische Aktivität, Inhibition, Anwendung in Synthesen

“…Diese Stereoinversion wurde kürzlich genutzt, um eine Methode zur Deracemisierung sekundärer Alkylsulfate zu entwickeln. [41][42][43] Auch die oxidative Spaltung von Alkylsulfatgruppen zu Aldehyden und anorganischen Sulfaten wurde beschrieben. [44][45][46] Gene für C-O spaltende [47,48] und oxidative Sulfatasen wurden kloniert.…”

“…[306,307] Weitere Anwendungen prokaryotischer Sulfatasen finden sich in der biologischen Sanierung von Alkylsulfatabfäl-len [48,308,309] und der Deracemisierung sekundärer Alkylsulfate. [41][42][43] …”

Highly Enantioselective sec‐Alkyl Sulfatase Activity of the Marine Planctomycete Rhodopirellula baltica Shows Retention of Configuration

“…Based on limited knowledge of the stereospecific and enantioselective hydrolysis of alkyl sulfate esters, [8] we recently reported an inverting alkyl sulfatase (termed "RS2") from Rhodococcus ruber DSM 44541. [10][11][12] Although the enzyme exhibited absolute stereospecificity through strict inversion of configuration of simple sec-alkyl sulfate esters, its enantioselectivity was less than perfect and its substrate tolerance was rather narrow. These limitations could be circumvented through the use of aerobically grown sulfur metabolizers.…”