Discovery of 3-formyl-tyrosine metabolites from Pseudoalteromonas tunicata through heterologous expression

Article abstract of DOI:10.1021/ja9097862

(Chemical Equation Presented) Genome mining and identification of natural product gene clusters typically relies on the presence of canonical nonribosomal polypeptide synthetase (NRPS) or polyketide synthase (PKS) domains. Recently, other condensation enzymes, such as the ATP-grasp ligases, have been recognized as important players in natural product biosynthesis. In this study, sequence based searching for homologues of DdaF, the ATP-grasp amide ligase from dapdiamide biosynthesis, led to the identification of a previously unannotated biosynthetic gene cluster in Pseudoalteromonas tunicata. Heterologous expression of the cluster in Escherichia coli allowed for the production and structure determination of two new 3-formyl tyrosine metabolites. Copyright

Full text of DOI:10.1021/ja9097862

Published on Web 12/30/2009
Discovery of 3-Formyl-Tyrosine Metabolites from Pseudoalteromonas tunicata
through Heterologous Expression
Leah C. Blasiak and Jon Clardy*
Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School, 240 Longwood AVe,
Boston, Massachusetts 02115
Received November 18, 2009; E-mail: Jon_Clardy@hms.harvard.edu
While chemists have long admired the remarkable and useful
set of molecules biosynthesized by bacteria, sequenced bacterial
genomes, which now appear at the rate of ∼20/month, reveal that
we actually know shockingly little about the repertoire of bacterially
produced small molecules. Cryptic metabolites, molecules whose
existence can be inferred from gene sequences but which have never
1-3
been seen in the laboratory, greatly outnumber known metabolites.
As a result, the discovery of cryptic molecules in genomes and
their production in the laboratory constitute a major research effort.
Most studies have focused on the nonribosomal polypeptide
synthetase (NRPS), polyketide synthase (PKS), or NRPS-PKS
hybrid pathways, as their large size and repeating motifs make them
easier to identify and analyze. Smaller pathways employing less
familiar biosynthetic strategies are easy to miss in genome
annotations, and small molecules made by these pathways are
doubly cryptic because they are detected neither in genome
annotations nor in the laboratory. These metabolites likely possess
new molecular templates and biological activities. This report
describes the search that led to the discovery of previously cryptic
metabolites that contain an unusual tyrosine-related amino acid (1
and 2) from Pseudoalteromonas tunicata.
We and others recently noted the ability of ATP-grasp-type
ligasessenzymes that catalyze condensation reactions by activating
a carboxyl group as a mixed phosphoric acid anhydride in primary
metabolismsto catalyze the formation of amide bonds in idiosyn-
cratically biosynthesized small molecules. Known examples include
amide bond formation in the posttranslational modification of the
4
,5
6
ribosomally synthesized molecules microviridin and bacilysin
and the amide bonds formed between nonproteogenic amino acids
7
in the dapdiamides. Sequence-based searching for homologous
ATP-grasp enzymes led to a promising biosynthetic gene cluster
in the marine gammaproteobacterium Pseudoalteromonas tunicata
D2, a model organism for microbial interactions on marine surfaces
and a known producer of a variety of antimicrobial and antifouling
Figure 1. (a) LC/MS traces of heterologous production of 1 and 2. (b)
LC/MS analysis showing ATP-dependent ligation of L-Thr and 2 by FtyB
to give 1. (c) Schematic of 3-formyl-L-tyrosine (fty) biosynthetic gene cluster
and proposed biosynthesis.
8
compounds.
To identify the products of the ATP-grasp containing gene
cluster, the predicted operon was PCR amplified from genomic
DNA, cloned into an inducible bacterial expression vector (pET-
Duet-1), and transformed into Escherichia coli. Culture supernatants
were fractionated and analyzed for the production of new com-
pounds by LC/MS and comparison to an empty vector control
correlations defined the 3-formyl tyrosine as the N-terminal amino
acid of dipeptide 1, and this linkage was verified by the ESI-LCQ-
MS/MS ion fragmentation pattern. Optical rotation showed that 2
9
was the L-enantiomer, and the absolute configuration of 1 was
determined by acid hydrolysis and Marfey’s derivatization of the
resulting amino acids.
(Figure 1a). Two new clone-specific peaks with molecular ions m/z
3
11 and 210 and absorption peaks at 340 nm were observed in
aqueous culture extracts. The corresponding compounds were
isolated and identified as 3-formyl-L-tyrosine-L-threonine dipeptide
(
1) and 3-formyl-L-tyrosine (2) by a combination of 1D and 2D
NMR including gCOSY, gHSQC, and gHMBC experiments (see
Supporting Information). The molecular formula of 1 was deter-
mined to be C14
[
H
19
N
2
O
6
by high resolution ESI-QToF-MS with
M+H]+ m/z 311.1232 (calc. m/z 311.1243). HMBC and COSY
9
26 9 J. AM. CHEM. SOC. 2010, 132, 926–927
10.1021/ja9097862  2010 American Chemical Society

C O M M U N I C A T I O N S
After discovery of these compounds through heterologous
expression, we returned to the native organism to find conditions
under which 1 and 2 could be produced. We detected 1 and 2 (∼1:2
ratio) in aqueous extracts of P. tunicata liquid marine broth cultures
the necessary elements for constructing 3-formyl-tyrosine. Detailed
biochemical investigation of the pathway is under way.
In summary, using a newly discovered role for ATP-grasp
enzymes as a search strategy for cryptic metabolites led to the
discovery of compounds 1 and 2. Placing their biosynthetic cluster
in an alternative and genetically manipulable host facilitated both
the production and detection of these compounds as well as a
preliminary interrogation of the individual biosynthetic genes. The
particular pathway found, the Fty pathway, also indicates that newly
discovered biosynthetic clusters will in turn suggest other genes,
such as FtyD and the FtyE/F pair, that can serve as the basis for
additional searches. A bioinformatic search for clusters containing
both FtyE and FtyF homologues revealed >20 unannotated clusters
(
see Supporting Information), and their identities were confirmed
by LC/MS and comparison to authentic standards (identical
retention times, UV-vis spectra, and molecular ion m/z values).
We initially flagged the fty (formyl tyrosine) cluster for further
study because of the ATP-grasp enzyme FtyB, which is homologous
(
30% identity) to an ATP-grasp-type amide ligase in dapdiamide
7
biosynthesis (DdaF). When a construct lacking FtyB was expressed
in E. coli only 2 was observed in culture extracts by LC/MS and
extracted ion traces, suggesting FtyB catalyzes amide bond forma-
tion between 2 and L-threonine to yield 1 (Figure 1a). To test this
16
as well as clusters for phenazine (EhpF/G from Erwinia herbicola)
and thienamycin (ThnN/O from Streptomyces cattleya).
1
7
hypothesis, a version of FtyB with an N-terminal His
6
tag was
cloned, expressed, and purified from E. coli (See Supporting
Information). FtyB activity was demonstrated using an LC/MS
based assay and o-phthalaldehyde (OPA) derivatization of substrates
and products. FtyB ligates L-Thr and 2 in an ATP-dependent fashion
to produce 1 (Figure 1b).
Most cryptic metabolites are cryptic because their production is
regulated, not constitutive, and their discovery leads to new
questions about the regulation of their production and their
biological function. In this regard, it is worth noting that compounds
1 and 2 exhibited no antimicrobial activity in agar diffusion assays
against E. coli, Bacillus subtilis, or Saccromyces cereVisae at doses
up to 20 µg/disk. However, similar synthetic compounds have
A bioinformatic analysis of the remaining enzymes in the cluster
suggests they are involved in the production of 2, the formylated
tyrosine. FtyE and FtyF are closely related to GriC and GriD (39%
and 27% identity respectively), which have been shown to function
as an aryl carboxylic acid reductase pair in grixazone biosynthesis
9
shown antihypertensive and appetite suppressant activities.
Acknowledgment. This work was supported by a grant from
the NIH to J.C. (RO1 GM086258). L.C.B. is the recipient of an
NIH NRSA postdoctoral fellowship (F32 GM087880). We thank
Sarah Mahlstedt for helpful discussions.
10
by Streptomyces griseus. FtyE/F could generate the formyl group
through conversion of a carboxylic acid to the acyl-AMP intermedi-
ate followed by NAD(P)H-dependent reduction. This is a common
route to aldehydes in nature; a well-known example is the
Supporting Information Available: Experimental details, charac-
terization data for new compounds, and table of sequence homologues.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
1
biosynthesis of the fatty aldehyde substrate of luciferase.
FtyD is homologous to deoxy-D-arabino-heptulosonate-7-phos-
phate (DAHP) synthase (30% identity with class II DAHP synthase
from Streptomyces coelicolor), the first step in bacterial chorismate
biosynthesis. DAHP synthase homologues are commonly present
in biosynthetic pathways that rely on the shikimate/chorismate
pathway from primary metabolism for substrates as expression of
a cluster specific DAHP synthase bypasses the key regulatory step
References
(1) Baltz, R. H. Curr. Opin. Pharmacol. 2008, 8 (5), 557–63.
(
(
(
2) Bode, H. B.; Muller, R. Angew. Chem., Int. Ed. 2005, 44 (42), 6828–46.
3) Hertweck, C. Nat. Chem. Biol. 2009, 5 (7), 450–2.
1
2
in chorismate production. One possible route to m-substituted
tyrosine derivatives from chorismate would be through isochoris-
mate and an isoprephenate-like intermediate. Isoprephenate has long
been suggested as an intermediate in the production of 3-carboxy-
substituted aromatic amino acids in plants but the responsible
4) Ziemert, N.; Ishida, K.; Liaimer, A.; Hertweck, C.; Dittmann, E. Angew.
Chem., Int. Ed. 2008, 47 (40), 7756–9.
5) Philmus, B.; Christiansen, G.; Yoshida, W. Y.; Hemscheidt, T. K.
ChemBioChem 2008, 9 (18), 3066–73.
(
(6) Steinborn, G.; Hajirezaei, M. R.; Hofemeister, J. Arch. Microbiol. 2005,
83 (2), 71–9.
1
(
7) Hollenhorst, M. A.; Clardy, J.; Walsh, C. T. Biochemistry 2009, 48 (43),
10467–72.
13,14
enzymes have never been identified.
FtyI contains a 300 residue
(
8) Thomas, T.; Evans, F. F.; Schleheck, D.; Mai-Prochnow, A.; Burke, C.;
Penesyan, A.; Dalisay, D. S.; Stelzer-Braid, S.; Saunders, N.; Johnson, J.;
Ferriera, S.; Kjelleberg, S.; Egan, S. PLoS ONE 2008, 3 (9), e3252.
domain homologous to isochorismate synthase (30%/62% identity/
similarity to E. coli MenF), and the NAD(P)-dependent oxidoreduc-
tase FtyH is a potential isoprephenate dehydrogenase candidate to
generate the aromatic ring.
(9) Kaiser, A.; Bretschneider, H.; Hohenlohe-oehringen, K. U.S. Patent 3896166,
1975.
(
10) Suzuki, H.; Ohnishi, Y.; Horinouchi, S. J. Antibiot. (Tokyo) 2007, 60 (6),
The aminotransferase homologue FtyJ (29% identity, 61%
similarity to Thermus thermophilus AspAT) likely converts the keto
acid to the amino acid. Heterologous expression of a construct
lacking FtyJ still afforded 1 and 2, although in 2-fold lower amounts,
which suggests that the activity of FtyJ can be complemented by
promiscuous E. coli aminotransferases (Figure 1a). FtyA is also
not required for heterologous production of 1 and 2. It has sequence
similarity to yeast COQ4 (27% identity), a protein implicated in
380–7.
(
(
11) Meighen, E. A. FASEB J. 1993, 7 (11), 1016–22.
12) Li, W.; Chou, S.; Khullar, A.; Gerratana, B. Appl. EnViron. Microbiol.
2
009, 75 (9), 2958–63.
(13) Larsen, P. O. Biochim. Biophys. Acta 1966, 115 (2), 529–31.
(
(
(
14) Zamir, L. O.; Nikolakakis, A.; Bonner, C. A.; Jensen, R. A. Bioorg. Med.
Chem. Lett. 1993, 3 (7), 1441–1446.
15) Marbois, B.; Gin, P.; Gulmezian, M.; Clarke, C. F. Biochim. Biophys. Acta
2
009, 1791 (1), 69–75.
16) Giddens, S. R.; Feng, Y.; Mahanty, H. K. Mol. Microbiol. 2002, 45 (3),
7
69–83.
1
5
ubiquinone biosynthesis but with no known enzymatic activity.
(17) Nunez, L. E.; Mendez, C.; Brana, A. F.; Blanco, G.; Salas, J. A. Chem.
Biol. 2003, 10 (4), 301–11.
While the order of the proposed biosynthetic reactions remains
unclear, bioinformatic analysis of the fty cluster suggests it contains
JA9097862
J. AM. CHEM. SOC. 9 VOL. 132, NO. 3, 2010 927