Scheme 1. Stereochemical Course of Retaining and Inverting Alkylsulfatases
ofsulfatasesis related to metallo-β-lactamasesand (to date)
harbors only a single member: Sodium dodecyl sulfatase
(SdsA1).6 Since SDS is an achiral prim-sulfate ester, the
stereochemical course of SdsA1-hydrolysis is not ‘visible’.
However, since the nucleophilic water molecule (W2 in the
crystal structure 2cfu) strongly interacts with the sulfur
atom of the substrate surrogate inhibitor 1-decanesulfo-
nate, retention at carbon was assumed.6
Inverting alkylsulfatases9 were previously studied in
Pseudomonas C12B,10 Comamonas terrigena,11 and Rhodo-
coccus ruber DSM 44541,12 but due to the lack of biochem-
ical and structural data the mechanism of inverting
alkylsulfatases remained unknown. In the search for a
well-characterized stable sec-alkylsulfatase, which would
allow the design of a deracemization process for sec-alcohols
via enantioconvergent hydrolysis of the corresponding sul-
fate esters, an extended whole-cell screening for sec-alkylsul-
fatase activity was conducted, which revealed Pseudomonas
sp. DSM 6611 as the most promising candidate.13
sequencing and peptide matching) surprisingly turned
out to be a homologue of SdsA1.15 The Pisa1 gene was
amplifiedbyPCR, cloned, and expressedinanE. coliBL21
strain with a C-terminal hexa-histidine tag. Pisa1 displayed
the desired catalytic properties: (R)-2-Octyl sulfate
(>99% ee) was quantitatively hydrolyzed to yield (S)-2-
octanol (>99% ee) through strict inversion of configura-
tion, whereas the (S)-enantiomer was completely unreac-
tive. Hydrolysis of rac-2-octyl sulfate ceased at 50%
conversion to furnish a homochiral product mixture of
(S)-2-octanol and unreacted (S)-2-octyl sulfate, indicating
perfect enantioselectivity (E >200).
Although Pisa1 shares a 44% sequence identity with
SdsA1 the substrate preference of both proteins differs
significantly: Based on kcat and KM values, SdsA1 has a
150-fold affinity for the prim-sulfate ester 1a, whereas
Pisa1 has a pronounced (190-fold) opposite preference
for the sec-sulfate ester analog 2a. Hence, Pisa1 is the first
inverting sec-alkylsulfatase that is characterized on a
molecular level.15,16
The stereochemical course of hydrolysis for both en-
zymes was investigated in detail using unlabeled 1-octyl-
(1a) and rac-2-octyl sulfate (2a) in 18O-enriched buffer
(label >98%, Scheme 2, part A). GC-MS analysis of the
formed 1-octanol (1b) and (S)-2-octanol (2b, ee >99%)
showed complete incorporation of the 18O-label in the
product within analytical limits, proving CꢀO bond clea-
vage in both cases.
The nucleophilic attack of (formal) [OHꢀ] at the chiral
carbon atom of an alkyl sulfate ester as exerted by alkyl-
sulfatases SdsA1 and Pisa1 leading to inversion of config-
uration at C is a remarkable catalytic event, which does not
have a direct counterpart in chemical catalysis. The latter
event would generate SO42ꢀ, which is a very poor leaving
group.17 Hence, invertingnucleophilichydrolysisofsulfate
Chromatographic protein purification followed by tryp-
tic digestion and peptide mass fingerprinting allowed
assignment of the obtained peptide masses to the predicted
open reading frames using the full genomic sequence of
the strain.14 The protein band with the lowest mobility
on SDS-PAGE (termed Pisa1, identified by de novo
(9) Bartholomew, B.; Dodgson, K. S.; Matcham, G. W. J.; Shaw,
D. J.; White, G. F. Biochem. J. 1977, 165, 575–580.
(10) Shaw, D. J.; Dodgson, K. S.; White, G. F. Biochem. J. 1980, 187,
181–196. White, G. F. Appl. Microbiol. Biotechnol. 1991, 35, 312–316.
(11) Fitzgerald, J. W.; Dodgson, K. S.; Matcham, G. W. J. Biochem.
J. 1975, 149, 477–480. Matcham, G. W. J.; Dodgson, K. S.; Fitzgerald,
J. W. Biochem. J. 1977, 167, 723–729. Barrett, C. H.; Dodgson, K. S.;
White, G. F. Biochem. J. 1980, 191, 467–473.
(12) Pogorevc, M.; Faber, K. Appl. Environ. Microbiol. 2003, 69,
2810–2815.
(13) Gadler, P.; Faber, K. Eur. J. Org. Chem. 2007, 5527–5530.
€
(14) Angelova, M.; Gadler, P.; Kayer, H.; Krempl, P.; Gully, C.;
Faber, K.; Macheroux P.; Thallinger, G. Manuscript in preparation.
(15) The sequence was submitted to the European Nucleotide Ar-
chive (accession #: FR850678 ꢀ confidential until 01.07.2011). No
measurable sequence similarities of Pisa1 with other sulfatases, such as
the R-ketoglutarate-dependent alkylsulfatase AtsK from Pseudomonas
putida, the arylsulfatase PAS from P. aeruginosa, and the human steroid
sulfatases HArsA, N-acetylgalactosamin 4-sulfatase, or cerebroside-3-
sulfate sulfatase, could be detected (e10%).
˚
(16) The coordinates of the crystal structure of Pisa1 (2.7 A
resolution) are available from the PDB (code 2YHE); A detailed study
on the structural and mechanistic aspects of Pisa1 will be published:
Knaus, T.; Schober, M.; Kepplinger, B.; Gadler, P.; Macheroux, P.;
Faber, K.; Wagner, U. G. Manuscript in preparation.
(17) Being the anion of the weak acid HSO4ꢀ (pKa = þ1.9 to þ2.7).
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