Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase purified from the detergent-degrading bacterium Pseudomonas C12B

Abstract

The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C 12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose, Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000-46 000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C-O bond of the C-O-S ester linkage was confirmed by 180-incorporation studies.

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter

“…Activity measurements were based on the amount of nitrite liberated. very significant component in the cellular protein, and this situation is similar to that obtained for other inducible enzymes (5,20,31).…”

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

“…4) suggests that the enzyme is nonglobular and thus indicates that the protein is monomeric. However, this enzyme appears to be much smaller than the previously described alkylsulfatases S1 from Pseudomonas strain C12B and CS2 from C. terrigena, which have molecular masses of approximately 250 kDa (2,21), and it seems to be more closely related to the inducible enzyme S3 from Pseudomonas strain C12B (molecular mass, 40 to 46 kDa) (26).…”

“…The first indication of the enantioselectivity of sec-alkylsulfatases was the chance observation that some racemic alkyl sulfates could be rapidly hydrolyzed to 50% conversion, whereas the remainder of the hydrolysis took place at a very reduced rate or not at all (20,26). In spite of this observation, only a single report on the enantioselectivity of alkylsulfatases has been published to date (33).…”

“…The preferred substrates for the enzyme were linear sec-alkyl sulfate esters, particularly 2-, 3-, and 4-octyl sulfates. Again, there was a certain similarity to inducible secondary alkylsulfatase S3 from Pseudomonas strain C12B, whose activity was centered on symmetrical alkylsulfate esters, such as 4-heptyl sulfate or 5-nonyl sulfate, as well as various nonsymmetrical C 3 -and C 4 -substituted alkyl sulfate esters (26). On the other hand, this is in contrast to the behavior of secondary alkylsulfatases S1 and S2 from Pseudomonas strain C12B and of CS2 from C. terrigena, which showed a striking affinity for 2-alkyl sulfates.…”

Comparison of pathways for biodegradation of monomethyl sulphate in Agrobacterium and Hyphomicrobium species

“…Longchain esters undergo hydrolysis catalysed by alkyl sulphatases in Pseudumonas sp. (Bartholomew et al, 1978;Bateman et al, 1986;Cloves et al, 1980;Dodgson & White, 1983;Dodgson et al, 1982;Matcham et al, 1977a, b ;Shaw et al, 1980;White & Russell, 1993) but these enzymes cease to function when the alkyl chain 0001-8396 0 1993 SGM length falls below C,. A coryneform species has been shown recently to produce an alkyl sulphatase restricted to primary esters in the C3-C7 chain length range (White & Matts, 1992).…”