New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo

Article abstract of DOI:10.1021/ja804530w

Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded aldehyde tag for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli-s FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins. Copyright

Full text of DOI:10.1021/ja804530w

Published on Web 08/23/2008
New Aldehyde Tag Sequences Identified by Screening Formylglycine
Generating Enzymes in Vitro and in Vivo
Jason S. Rush and Carolyn R. Bertozzi*
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute,
UniVersity of California, Berkeley, California 94720
Received June 14, 2008; E-mail: crb@berkeley.edu
Formylglycine generating enzyme (FGE) was identified in 2003
as the posttranslational machinery that activates type I sulfatases
in eukaryotes.1 The enzyme oxidizes a cysteine residue within a
∼13 amino acid consensus sequence, also termed the “sulfatase
Figure 1. Reaction catalyzed by FGE.
motif”, forming an aldehyde-bearing formylglycine (FGly) residue
(Figure 1) that is critical for the sulfatases’ catalytic function.2 In
eukaryotes, FGE requires a minimal submotif, CxPxR,3,4 that is
highly conserved among all type I sulfatases. However, in prokary-
otes either CxP/AxR5 motifs or serine-based SxPxR6 motifs are
found within sulfatases. Prokaryotic FGEs, first characterized from
Mycobacterium tuberculosis and Streptomyces coelicolor,7 recog-
nize CxPxR, while anaerobic sulfatase-maturating enzymes (anS-
MEs) act on both CxAxR and SxPxR.5 FGEs and anSMEs have
distinct sulfatase substrates and catalytic mechanisms.7,8
In addition to its intriguing biological function, FGE has also
attracted attention as a tool for protein engineering. Conversion of
cysteine to FGly introduces a uniquely reactive aldehyde group at
Figure 2. A high-throughput assay for FGE activity.
a specific site dictated by the sulfatase motif. Recently, we reported
that a six-residue sulfatase submotif (LCTPSR) can be introduced
into heterologous proteins while maintaining in ViVo conversion to
FGly during expression in E. coli.9 Once the aldehyde group was
posttranslationally installed, chemoselective ligation with aminooxy-
or hydrazide-functionalized molecules enabled site-specific protein
modification. We employed this genetically encoded “aldehyde tag”
for site-specific labeling of proteins with probes and polyethylene
glycol (PEG) groups.
Although the six-residue aldehyde tag is a relatively small motif,
its foreign sequence may perturb local structure or confer immu-
nogenicity on therapeutic proteins. These potential liabilities
prompted us to focus on expanding the repertoire of aldehyde tag
sequences, with the ultimate objective of designing motifs that
minimally perturb the host protein. Perusal of bacterial genomes
that encode putative FGEs revealed sulfatase submotifs that differ
from the canonical sequence CxPxR.10 Therefore, naturally occur-
ring FGEs might recognize a spectrum of motifs that could serve
as diverse aldehyde tags for protein engineering.
In this work, we probed the specificities of FGEs from M.
tuberculosis and S. coelicolor using an alanine-scanning peptide
substrate library. We developed an in Vitro assay (Figure 2) that
monitors conversion of cysteine to FGly within synthetic N-
terminally biotinylated peptide substrates. The peptides were first
incubated with FGE, after which the newly formed aldehydes were
reacted with aminooxy-functionalized 2,4-dinitrophenyl (2,4-DNP)
conjugate 1.11 The resulting oxime-linked products were captured
on NeutrAvidin-coated microtiter plates. Colorimetric detection was
accomplished by incubation with a commercial anti-2,4-DNP anti-
body conjugated to alkaline phosphatase (R-2,4-DNP-AlkPhos)
followed by treatment with p-nitrophenyl phosphate (pNPP).12
We generated two peptide libraries based on 13-residue motifs
found in putative sulfatases from the two prokaryotes (ICT-
PARASLLTGQ and LCTPSRGSLFTGR, from S. coelicolor and
M. tuberculosis, respectively). Each residue within the sequences
was probed by alanine substitution to generate a total of 28 peptides
including the two wild-type sequences (native alanine residues
within the S. coelicolor sequence were substituted with glycine).
The percent conversion of cysteine to FGly was quantified for each
alanine- (or glycine)-substituted peptide relative to that of the
corresponding wild-type sequence.
As shown in Figure 3, the two FGEs displayed different tol-
erances for alanine mutations within the sulfatase motifs. Substitu-
tion at any position in the native sequence recognized by S.
coelicolor FGE resulted in significant reduction in FGly formation
(Figure 3a, blue bars). Replacement of Thr3, Pro4, Arg6, or Leu9
with alanine was particularly detrimental. A similar specificity
profile was observed with the library derived from the M.
tuberculosis sulfatase motif (Figure 3b). Human FGE, which has a
51% amino acid sequence identity to S. coelicolor FGE, also has
a strict requirement for Pro and Arg within the CxPxR sequence.3,4
However, the human enzyme is known to tolerate substitutions
corresponding to Thr3 or Leu9,4 indicating species-specific variation
in substrate preference. Surprisingly, M. tuberculosis FGE displayed
a much greater tolerance for alanine substitution in both sulfatase
motif libraries (Figure 3a and b, red bars). Notably, replacement
of Pro4 or Arg6 with alanine was well tolerated, as were
substitutions in the C-terminal region.
Despite the 46% amino acid sequence identity shared by M.
tuberculosis and S. coelicolor FGEs, their response to alanine
substitutions in peptide substrates is very different. To gain insight
into the molecular basis of substrate discrimination, we generated
structural models of FGE-peptide complexes using the S. coelicolor
enzyme’s crystal structure7 and a homology model of M. tubercu-
losis FGE (Figure 4).14 These models indicated that the substrate’s
conserved Pro residue binds within a pocket that varies considerably
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10.1021/ja804530w CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 5. SDS-PAGE of MBP constructs bearing the C-terminal aldehyde
tag sequences shown above each lane. The proteins were expressed in E.
coli, purified on Ni-NTA spin columns, and reacted with Alexa Fluor 647
C5-aminooxyacetamide (Aminooxy Alexa Fluor 647). Fluorescence images
of the gel are shown. Top, Alexa Fluor 647. Bottom, protein loading as
determined by Sypro Orange.
The E. coli machinery converted all three sequences testeds
LCTPSR (wild-type), LCTASR, and LCTASAsat comparable
levels, while no signal was observed for any of the C-to-A mutants.
Alanine substitution of the conserved Pro and Arg residues within
the canonical sequence did not significantly reduce conversion
efficiency. This striking observation suggests that a wide range of
aldehyde tag sequences are recognized in E. coli, offering a practical
system for expression of modified proteins.
In summary, peptide library screening revealed noncanonical
sequences that are recognized by M. tuberculosis FGE in Vitro and
the E. coli FGE-like activity in ViVo. This finding expands the range
of aldehyde tag sequences for protein engineering. An important
future goal is to identify the molecular nature of E. coli’s machinery.
Figure 3. FGE activity on peptide substrates. (a) Relative activity of S.
coelicolor (blue) and M. tuberculosis (red) FGEs on peptides derived from
the S. coelicolor sulfatase motif. (b) Relative activity of S. coelicolor (blue)
and M. tuberculosis (red) FGEs on peptides derived from the M. tuberculosis
sulfatase motif. Error bars represent the standard deviation of three replicates.
Acknowledgment. We thank M. Breidenbach and B. Carlson
for technical assistance. This work was supported by grants to
C.R.B. from the National Institutes of Health (GM059907 and
Nanomedicine Development Center).
Supporting Information Available: Experimental procedures,
spectral data, and assay data. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 4. Models of prokaryotic FGE active sites with peptide substrate
bound. (a) Crystal structure of S. coelicolor FGE with modeled peptide
substrate. (b) Homology model of M. tuberculosis FGE with modeled
peptide substrate. The substrate peptide shown in cyan is CTPSR. Colors
indicate electrostatic potential (blue, positive; red, negative).
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(11) See Supporting Information for synthetic details.
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spectrometry. See Supporting Information for details.
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