Highly enantioselective sec-alkyl sulfatase activity of the marine planctomycete Rhodopirellula baltica shows retention of configuration

Article abstract of DOI:10.1002/anie.200501955

(Chemical Equation Presented) Hydrolytic enzymes: The marine planctomycete Rhodopirellula baltica DSM 10527 displays high stereo- and enantioselective alkyl sulfatase activity towards (±)-sec-alkyl sulfates with retention of configuration through cleavage of their S-O bond (see scheme; pathway B), whereas inversion of configuration is observed upon cleavage of the C-O bond (pathway A).

Full text of DOI:10.1002/anie.200501955

Angewandte
Chemie
Sulfatases
DOI: 10.1002/anie.200501955
Highly Enantioselective sec-Alkyl Sulfatase
Activity of the Marine Planctomycete
Rhodopirellula baltica Shows Retention of
Configuration**
Sabine R. Wallner, Marga Bauer, Chris Würdemann,
Patricia Wecker, Frank O. Glöckner, and Kurt Faber*
The molecular mechanisms of the commonly employed
hydrolases involve nucleophilic attack onto the carbonyl
group of carboxylic acids or their derivatives.[1,2] As this group
is a planar entity, any stereochemical alterations of the
substrate caused by enzymatic catalysis are impossible, and as
a consequence, enantiomers of the transformed substrate and
product are usually homochiral (with the exception of
prochiral or meso esters), that is, they have the same absolute
configuration. Although the stereochemical features of the
substrate, such as stereogenic centers (in racemates) or
enantiotopic groups (in prochiral or meso compounds), are
“recognized” by the enzyme, which gives rise to differences in
kcat and/or KM values, they remain unchanged during catalysis.
Biocatalysts, which elicit the more complex potential to
affect the stereochemistry of the substrate in a controlled
fashion during catalysis, are rather rare and encompass
haloalkane dehalogenases,[3] epoxide hydrolases,[4] and
(alkyl) sulfatases.[5,6] In each case, a C(sp3) atom could
potentially be involved in the catalysis and therefore open
the possibility of stereocomplementary pathways. These
enzymes do not only display enantioselectivity (through the
transformation of one substrate enantiomer faster than the
other) but also stereoselectivity (with retention or inversion
of configuration). This therefore makes them important
catalytic tools for the development of so-called enantiocon-
vergent processes in which each enantiomer from a racemic
mixture is transformed into the same product through
independent pathways, that is, through retention and inver-
sion of configuration.[7] As a consequence, a racemate can be
[*] Dr. S. R. Wallner, Prof. K. Faber
Research Centre for Applied Biocatalysis
Department of Chemistry, Organicand BioorganicChemistry
University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
Fax: (+43)316-380-9840
E-mail: Kurt.Faber@Uni-Graz.at
Dr. M. Bauer, C. Würdemann, P. Wecker, Dr. F. O. Glöckner
MPI Marine Microbiology
Celsiusstrasse 1, 28359 Bremen (Germany)
[**] This study was performed within the Research Centre for Applied
Biocatalysis. Financial support by the FFG, the City of Graz, and the
Province of Styria is gratefully acknowledged. L. Arbanas, T. Glieder,
W. Kroutil, and U. Wagner (Graz) are thanked for their valuable
contributions in sequence analysis.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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converted, in principle, into a single stereoisomeric product
was elucidated during structural studies on human aryl
without the occurrence of an undesired stereoisomer.
Sulfatases catalyze the hydrolytic cleavage of the sulfate
ester bond (Scheme 1). Depending on the enzyme subtype,
the stereochemical course of sulfate ester hydrolysis can be
sulfatase A[16] and was shown to proceed through cleavage
[17]
À
of the S O bond. The latter is effected by nucleophilic
attack of an aldehyde hydrate (formed from a Cys or Ser
residue by post-translational modification[18]) onto the sulfur
atom; the result is liberation of the
corresponding alcohol and retention of
its stereochemistry (Scheme 2).[19]
The same principle of catalysis was
shown to occur in sulfatases from Pseu-
domonas aeruginosa[20] and Klebsiella
pneumoniae.[21] As the natural sub-
Scheme 1. Enzymaticstereodivergent hydrolysis of sulfate esters catalyzed by sulfatases.
strates of these enzymes are believed
to be glycosyl and aryl sulfate esters,[16]
controlled by an appropriate enzyme. On the one hand,
nothing is known about their enantioselectivities. Our search
for a suitable microbial source for retaining (alkyl) sulfatase
activity was led by the fact that all known sulfatases that act
through the above-mentioned mechanism have the canonical
sulfatase consensus motif C/S-X-P-X-R-X4-T-G.[18,22,23,27] This
sequence motif encompasses sulfatases from mammals, lower
eukaryotes, and prokaryotes,[24] but is absent in the inverting
alkyl sulfatase RS2 from Rhodococcus ruber.[25]
A recently published complete genome sequence of the
marine planctomycete Rhodopirellula baltica DSM 10527
(formerly denoted as Pirellula sp. 1)[26,27] revealed the exis-
tence of an unexpectedly high number of 110 genes putatively
encoding sulfatases. It is believed that, in R. baltica, these
enzymes play a vital role in the metabolism of sulfated
carbohydrates, such as chondroitin. Of the 110 potential
sulfatase sequences, 85 show the conserved “core” of the
sulfatase consensus motif C-X-P-X-R, which is required in
post-translational modification to render activated sulfatase
proteins. Furthermore, each of the 85 sequences contains the
adjacent “auxiliary” part of the consensus motif, X4-T-G,
either fully conserved or only slightly modified. This part of
the sulfatase consensus motif has been implicated in modu-
lating the efficiency of the post-translational modification
step.[24] According to the sequence–function relationships
proposed for both eukaryotic and prokary-
inverting sulfatases[8,9] were shown to act on sec-alkyl sulfate
À
esters by breaking the C O bond of the sulfate ester, which
resulted in inversion of configuration (Scheme 1, pathway A).
However, nothing is known about their mechanism of action.
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.[10–12] 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. In particular, Sulfolobus spp., which exhibited
not only enhanced enantioselectivities but also a broader
substrate spectrum,[13] showed a stereoselectivity identical to
that of “RS2”, that is, inversion of configuration was
observed.
To exploit the full potential of the enzymatic hydrolysis of
alkyl sulfate esters, stereocomplementary sulfatases that act
with retention of configuration were needed. In contrast to
inverting sulfatases, the mechanism of action of retaining
sulfatases is well understood (Scheme 2).[14,15] The mechanism
otic sulfatases,[20,22,24] the 85 putative sulfa-
tases from R. baltica were expected to act
through retention of configuration.
To prove this hypothesis, a set of sec-
alkyl sulfate esters, rac-1a–9a and the
primary sulfate ester 10a were screened
by using resting whole cells of R. baltica
DSM 10527 (Table 1). In general, the enan-
tiopreference was shown to be R, that is, R
enantiomers were preferentially hydro-
lyzed from the racemate, while S enan-
tiomers remained untouched.
R. baltica displayed excellent activities
on linear (w-1)-sulfate esters rac-1a–3a
(Table 2, entries 1–3), which were readily
accepted as substrates. Surprisingly, even
sterically demanding sulcatyl sulfate (rac-
4a), which was accepted by neither sulfa-
tase RS2 nor Sulfolobus spp., was con-
Scheme 2. Schematic representation of the mechanism of action of retaining (aryl) sulfatases.
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
verted with good rates. Concerning the
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Angewandte
Chemie
Table 1: Enantioselective hydrolysis of sec-alkyl sulfates with retention of
configuration.
without racemization in > 99% ee; analogously, enantiopure
(S)-2-octyl sulfate remained untouched. Screening of the
extracellular medium did not show any activity, which
suggests that these sulfatases are intracellular. Using R.
baltica grown on various C sources—glucose, chondroitin
sulfate A,[28] and chondroitin sulfate C—did not lead to
significant differences in either activity or selectivity, thus
indicating that no enzyme induction takes place. This suggests
that the observed activity can, most likely, be associated with
a “broad-spectrum sulfatase” rather than with separate
primary- or sec-alkyl sulfatases.
In summary, the first highly enantioselective sec-alkyl
sulfatase that acts with strict retention of configuration was
detected in R. baltica DSM 10527 through a sequence-sim-
ilarity approach. The use of this sulfatase in combination with
a stereocomplementary inverting enzyme from Sulfolobus
spp. in a deracemization strategy is currently being inves-
tigated.[29]
Compound
R1
R2
1a,b
2a,b
3a,b
CH3
CH3
CH3
n-C6H13
n-C5H11
n-C7H15
=
4a,b
CH3
CH3
CH3
n-C2H5
n-C3H7
(CH3)2C CH(CH2)2
PhCH2
5a,b[a]
6a,b[a]
7a,b
Ph(CH2)2
n-C5H11
8a,b
n-C4H9
n-C5H11
n-C7H15
Received: June 6, 2005
Published online: September 14, 2005
9a,b[b]
10a,b
CH2 CH
=
H
[a] No conversion. [b] Opposite absolute configuration due to a switch in
Cahn—Ingold priority (CIP) assignment of R1 and R2.
Keywords: enantioselectivity · hydrolysis · stereoselectivity ·
sulfatases · sulfate esters
.
Table 2: Hydrolysis of sulfate esters using glucose-grown Rhodopirellula
baltica DSM 10527.
[1] This may be an ester, amide, lactone, hydantoin, lactam, or some
thio derivative.
Entry Substrate Conversion Product ee
Enantioselectivity
[2] U. T. Bornscheuer, R. J. Kazlauskas, Hydrolases in Organic
Synthesis, Verlag Chemie, Weinheim, 1999.
[%]
[%]
(E value)
1
2
3
4
5
6
7
8
9
rac-1a
rac-2a
rac-3a
rac-4a
rac-5a
rac-6a
rac-7a
rac-8a
rac-9a
10a
16
13
18
11
n.c.
n.c.
3
10
6
26
(R)-1b
(R)-2b
(R)-3b
(R)-4b
–
>99 >200
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>99 >200
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–
–
3
2
3
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(R)-7b
(R)-8b
(S)-9b[a]
10b
48
29
42
n.a.
10
n.a.
[a] Absolute configuration is S owing to a switch in CIP priority rules,
however, it is homochiral to (R)-1b–4b, 7b, 8b. n.c.=no conversion;
n.a.=not applicable. E values were calculated from E=
{ln[1Àc(1+eep)]}/{ln[1Àc(1Àeep)]} where c=conversion and eep =en-
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enantioselectivities, all four substrates (Table 2, entries 1–4)
showed perfect E values (> 200) and the corresponding
R alcohols 1b–4b were obtained with excellent ee values (>
99%). However, in the case of phenyl-substituted substrates
(Table 2, entries 5 and 6), no conversion was observed. As R1
and R2 became similar in size, yielding near-symmetrical
compounds (Table 2, entries 7–9), the enantioselectivities
decreased as was observed in previous studies on inverting
sulfatases.[9,10,13] As R. baltica has more than 100 genes that
encode putative sulfatases, it is not surprising that the primary
sulfate ester 10a was readily converted as well.
Stereochemical analysis of the products suggested that the
hydrolysis pathway proceeded with retention of configura-
tion. For unambiguous proof, enantiopure (R)-2-octyl sulfate
was used as the substrate, which yielded (R)-2-octanol
Angew. Chem. Int. Ed. 2005, 44, 6381 –6384
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6383

Communications
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[28] Chondroitin sulfate A bears a sec-sulfate ester (at position 4 on
the sugar moiety), whereas chondroitin sulfate C bears a primary
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[29] For a preliminary communication on the proof of principle, see
reference [6].
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