Identification and characterization of the first ovothiol biosynthetic Enzyme

Article abstract of DOI:10.1021/ja109378e

Ovothiols are histidine-derived thiols that were first isolated from marine invertebrates. We have identified a 5-histidylcysteine sulfoxide synthase (OvoA) as the first ovothiol biosynthetic enzyme and characterized OvoAs from Erwinia tasmaniensis and Trypanosoma cruzi. Homologous enzymes are encoded in more than 80 genomes ranging from proteobacteria to animalia.

Full text of DOI:10.1021/ja109378e

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Identification and Characterization of the First Ovothiol Biosynthetic
Enzyme
Andrea Braunshausen and Florian P. Seebeck*
Abteilung Physikalische Biochemie, Max Planck Institut f€ur Molekulare Physiologie, Otto-Hahn Strasse 11, 44227 Dortmund, Germany
S Supporting Information
b
EgtB from Mycobacterium smegmatis (E < 3 Â 10-7). EgtB and
ABSTRACT: Ovothiols are histidine-derived thiols that
were first isolated from marine invertebrates. We have
identified a 5-histidylcysteine sulfoxide synthase (OvoA)
as the first ovothiol biosynthetic enzyme and characterized
OvoAs from Erwinia tasmaniensis and Trypanosoma cruzi.
Homologous enzymes are encoded in more than 80 gen-
omes ranging from proteobacteria to animalia.
the putative OvoA share a domain with homology to formylglycine-
generating enzymes and an uncharacterized N-terminal domain.
In addition, the trypanosomatid protein contains a C-terminal
putative methyltransferase domain that appears to be specific for
ovothiol biosynthesis because ergothioneine-producing organ-
isms lack proteins with significant homology. Using this addi-
tional domain as a criterion to distinguish OvoAs from EgtBs, we
identified more than 80 OvoA homologues, predominantly from
proteobacteria but also from uni- and multicellular eukaryotes
(Figure S2 in the Supporting Information).
For in vitro characterization, we prepared recombinant OvoA
from E. tasmaniensis15 (OvoAe) and T. cruzi (OvoAt). Production
in Escherichia coli and purification on Ni-NTA agarose yielded
5 mg/L OvoAe but less than 0.5 mg/L OvoAt (Figures S3-S5).
To assess the catalytic activity, OvoAe was incubated with 1 mM
histidine, 3 mM cysteine, 2 mM TCEP, 0.1 mM FeSO4, 20 mM
Tris (pH 8.0), and 20 mM NaCl at 26 °C. Cation-exchange
HPLC analysis of this reaction revealed consumption of histidine
and production of a new compound that was identified as 2 by mass
spectrometry and NMR spectroscopy (HRMS: calcd. 291.07584,
found 291.07577; Figures S6 and S7). A single proton signal in
the aromatic range (C2-H: s, δ 8.1) is consistent with attach-
ment of the sulfoxide group to the imidazol C5. To confirm this
interpretation, we repeated the above experiment using C2-2H
histidine as the substrate and found that the corresponding product
retained the isotopic label (HRMS: calcd 292.08204, found
292.08215; Figure S7). The preference for C5 versus C2 oxida-
tion is the prime difference between OvoA and EgtB. Unlike 1,
the 2-thioimidazol ring of 3 exists predominantly in its thione
form, which is less acidic (pKa,SH > 10), less nucleophilic, and
markedly less prone to auto-oxidation than 1.16 It is quite likely
that these differences translate into distinct biological roles for
1 and 3.
On the basis of HPLC analysis, we determined that OvoAe
produces 2 with a rate constant of 1.9 ( 0.2 min-1, catalyzing at
least 140 turnovers per active site (Figures S8 and S9). In comparison,
the trypanosomal enzyme is significantly less active and less stable
(Figure S10). The in vitro activities of both enzymes are modest,
which might point to suboptimal assay conditions. For example,
cysteine and/or histidine may not be the true substrates. Therefore,
we assayed OvoAe with numerous thiols, such as glutathione and
γ-glutamylcysteine, and with π-N-methylhistidine, R-N-methyl-
histidine, and R-N,N-dimethylhistidine but found no superior
substrates (Figures S11-S15). Other factors, such as missing
vothiol A (1, Figure 1), B, and C are π-N-methyl-5-
O
thiohistidines with a very acidic thiol group (pKa = 1.4),1
proficiency as a one-electron donor,2 and a redox potential (-0.09 V
vs NHE) rivaling that of protein disulfide isomerases.1,3 These
properties render ovothiols efficient scavengers of radicals and
peroxides with possible roles in the redox defense of a number of
organisms.2,4-6 For example, sea urchin eggs contain millimolar
concentrations of ovothiol C, which protect the egg content
during oxidative envelope maturation.7 More recent reports that
pathogenic Trypanosoma and Leishmania produce 1 raised the
interest in its biochemistry and biosynthesis.4,5,8 Cell-free ex-
tracts from Crithidia fasciculata revealed that assembly of 1 starts
with the conversion of cysteine and histidine to a 5-histidylcys-
teine sulfoxide conjugate (2) in an oxygen-dependent reaction.
This intermediate is then trimmed to 5-thiohistidine and methy-
lated at the imidazole ring (Figure 1).4,9,10
Insertion of a sulfur atom into a nonelectrophilic, aromatic
C-H bond is quite unusual, and the involvement of oxygen
suggests a departure from known mechanisms for enzymatic
C-S bond formation, which are either oxygen-independent or
oxygen-sensitive.11-13 Therefore, deciphering the enzymology
of oxidative sulfur transfer presents an important challenge. Also,
the physiological functions of 1 are poorly understood, and the
potential of ovothiol biosynthesis as a target for anti-infective
agents is untested. To enable such research, we have character-
ized the first ovothiol biosynthetic enzyme from Erwinia tasma-
niensis and Trypanosoma cruzi.
We previously identified a mycobacterial enzyme (EgtB) that
inserts the sulfur atom of γ-glutamylcysteine into the C2-H bond
of histidine betaine to form an intermediate of the ergothioneine
biosynthetic pathway (3, Figure 1).14 On the basis of the
similarity of this reaction with the first step in ovothiol biosynthesis,
we surmised that distant EgtB homologues encoded in genomes
of ovothiol producers5 might be 5-histidylcysteine sulfoxide
synthases (OvoAs). In agreement with this idea, all five sequenced
trypanosomatid genomes contain genes with significant similarity to
Received: October 25, 2010
Published: January 19, 2011
r
2011 American Chemical Society
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histidine. Similarly, many non-heme iron enzymes depend on
R-ketoglutarate as an electron donor.19 For example, taurine
hydroxylase oxidizes R-ketoglutarate to succinate and carbon
dioxide to produce an iron(IV)-oxoiron species that abstracts a
hydrogen atom from a methylene group of taurine.20 The
resulting carbon radical recombines with an iron-bound hydroxyl
radical to form hydroxytaurine. An analogous OvoA mechanism
would require the formation of an sp2 radical (b in Figure 2).
Such species are highly unstable, making hydrogen atom transfer
a good candidate for the rate-limiting step. Despite this expecta-
tion, we could not detect any kinetic isotope effect in a competi-
tion between histidine and C-2,5,R-2H-histidine for OvoAe-
catalyzed turnover (Figure S18). Clearly, more experimental
work is necessary to examine this mechanistic proposal and
investigate alternative pathways, such as via the formation of a
histidyl π-radical or via electrophilic attack of iron-coordinated
cysteine sulfoxide on histidine (c and d, respectively, in Figure 2).
This report has characterized OvoA, the first enzyme in
ovothiol biosynthesis. Our data show that the enzyme requires
coordination of iron(II) to an unusual iron-binding motif.
Cysteine and histidine are preferred OvoAe substrates, but the
modest in vitro activities may suggest that the recombinant enzymes
are not fully active. To our knowledge, OvoA is the first character-
ized protein that mediates histidine side-chain modification at
C5. The identification of OvoA sets the stage for the construction
of ovothiol-deficient trypanosomatids to test whether this bio-
synthetic pathway is a target for novel anti-infective therapeutics.
Figure 1. Biosynthesis of ovothiol A (1) and ergothioneine (3). We
have adopted IUPAC histidine nomenclature to name ovothiol deriva-
tives (in red). Ovothiols B and C are the R-N-methyl and R-N,N-
dimethyl derivatives of ovothiol A; OvoA and EgtB are sulfoxide
synthases involved in ovothiol and ergothioneine biosynthesis.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures S1-S18, Table S1,
b
detailed experimental procedures, and 1H and 2D NMR spectra.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Figure 2. (left) HPLC analysis of enzymatic reactions of wild-type
OvoAe, iron-depleted wild-type OvoAe, and OvoAe variants H170A,
H174A, and E176A. HPLC signals were identified by ESI-MS. (right)
Proposed mechanisms for OvoA: C-S bond formation is initiated by
(b) formation of a histidyl sp2 radical, (c) formation of a histidyl π-
radical, and (d) electrophilic attack of the iron-coordinated cysteine
sulfoxide on histidine.
Corresponding Author
florian.seebeck@mpi-dortmund.mpg.de
’ ACKNOWLEDGMENT
We thank R. S. Goody for financial support, S. Gentz for technical
assistance, B. Griewel and F. Hofmann for NMR measurements,
T. Seebeck for trypanosomal DNA, and S. Sasso and K. J.
Woycechowsky for helpful comments.
post-translational modifications, absent protein complex part-
ners, or suboptimal pH and buffer composition, may limit the in
vitro activities of the recombinant enzymes.
OvoA and EgtB are both iron-dependent enzymes (Figure 2
and Figure S16).14 The molecular basis for iron recognition
probably maps to a conserved HX3HXE motif in the N-terminal
domains of both OvoA and EgtB (Figure S17). Consistent with
this thought, we found that removing any one of the three side
chains of His170, His174, and Glu176 reduces the OvoAe activity
by at least 100-fold (Figure 2). Although 2-His-1-carboxylate
facial triads are common iron-binding motifs,17 the proximity of
the ligands in the primary sequence of OvoA is unusual. An
alternative protein fold or an atypical geometry at the iron center
may necessitate this compact arrangement.
One further puzzle is why OvoA and EgtB couple C-S bond
formation to sulfoxidation,10,14,18 a modification that is not necessary
for subsequent biosynthetic steps and is absent in the final product.
Possibly, OvoA oxidizes cysteine to access an iron(IV)-oxo state
(a in Figure 2) which then mediates oxidative sulfurization of
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