(2S,3R)-3-Methyl Glutamate Biosynthesis
A R T I C L E S
coelicolor and the A54145 lipopeptide from Streptomyces
fradiae are produced as mixtures of compounds containing
In this study, the methyltransferase GlmT from Streptomyces
coelicolor is biochemically characterized in vitro enabling a
complete stereospecific in vitro synthesis of (2S,3R)-3-MeGlu
using S. coelicolor enzymes exclusively. Furthermore, we show
that DptI and LptI from S. roseosporus and S. fradiae catalyze
the same reaction as GlmT. By testing various possible
substrates, we determine the substrate specificity of the meth-
yltransferase. This combined with recent in vivo results19
provides complete mechanistic details of the individual steps
occurring during the biosynthesis and incorporation of the
1
3,17
3
-MeGlu or Glu,
fermentative production of daptomycin
from Streptomyces roseosporus results exclusively in 3-MeGlu
containing peptides.18 For daptomycin and CDA, this residue
1
9-21
was shown to be the (2S,3R)-stereoisomer of 3-MeGlu.
In
all of the acidic lipopeptides tested so far, the 3-MeGlu-
containing products were shown to exhibit a higher bioactivity
than the Glu-containing analogues.1
4,16,18,19,22
These observations identify this building block as a promising
target for engineering acidic lipopetide biosynthesis controlling
the incorporation of Glu, 3-MeGlu, or further Glu analogues in
search for improved bioactivities. However, from the chemical
point of view, the stereospecific functionalization of amino acids
at the relatively unreactive â-position is a synthetic challenge.
Despite this, there are some reports concerning asymmetric
3-MeGlu residues into the acidic lipopeptide group of antibiotics.
Notably the study presents the first example of enzymes from
secondary and primary metabolism cooperating in the biosyn-
thesis of a nonproteinogenic amino acid required for assembly
of nonribosomal peptides. It is also possible that similar
pathways, utilizing both primary and secondary metabolic
enzymes, may exist in the biosynthesis of other â-methylated
amino acid building blocks, such as the â-methylated Phe
2
3-25
synthesis of 3-MeGlu diastereoisomers,
convergent multistep synthesis to all possible stereoisomers of
-MeGlu was published.19 However, the in vitro utilization of
and, recently, a
2
6
residue of the mannopeptimycins.
3
the biosynthetic enzymes responsible for 3-MeGlu formation
would enormously facilitate the preparation of 3-MeGlu and
other â-functionalized Glu analogues, which are required for
engineering new lipopeptide products. For these reasons, it is
crucial to understand the biosynthesis of 3-MeGlu and to
determine the mechanism of its incorporation into the acidic
lipopeptide backbone.
Experimental Section
Strains, Culture Media, and General Methods. The E. coli strains
were grown in Luria-Bertani medium, supplemented with 100 µg/
mL ampicillin (final concentration). Oligonucleotides were purchased
from Operon. DNA dideoxy sequencing confirmed the identity of all
plasmids constructed.
Cloning and Expression of Glmt, IlvE, CDAPS3-PCP10, DptI,
and LptI. The genes coding for glmT, ilVE, and the cdaPS3-PCP10
gene fragment were amplified by PCR from chromosomal DNA of S.
coelicolor A3(2) (DSM 40783) using the Phusion polymerase
A gene SCO3215 was identified within the CDA biosynthetic
gene cluster of S. coelicolor, which was suggested to encode a
S-adenosyl methionine (SAM)-dependent glutamate-3-methyl-
transferase (GlmT) based on low, but significant, sequence
(Finnzymes). The genes coding for dptI and lptI were amplified from
S. roseosporus (NRRL 11379) and from S. fradiae (NRRL 18158) using
the same method. According to the manufacturer’s protocol for PCR
amplification of template DNA with high GC-content (S. coelicolor
74%), the dNTP concentrations were increased to 20 mM. Amplification
of glmT (SCO3215) was carried out using the oligonucleotides 5′-glmT
9
similarity to other methyltransferases. Within the daptomycin
and A54145 gene clusters in S. roseosporus and S. fradiae, there
also exist genes dptI and lptI encoding proteins that show high
similarity to GlmT. Fermentation of deletion mutants of S.
coelicolor (∆glmT) and S. roseosporus (∆dptGHIJ) led to
production of CDA and daptomycin analogues, respectively,
containing exclusively Glu instead of 3-MeGlu.1 Moreover,
complementation of the ∆dptGHIJ mutant by dptI or glmT and
complementation of ∆glmT with synthetic 3-MeGlu restored
(5′-AAA AAA CCA TGG TGA CCG GGG ACG ACG TGC AGG
GG) and 3′-glmT (5′-AAA AAA AAG CTT TGC CGC CTT CCC
GGC GGT GGC CG). The ilVE gene (SCO5523) was amplified using
the primer combination of 5′-ilVE (AAA AAA GGA TCC ATG ACG
ACG CCC ACG ATC GAG CTC) and 3′-ilVE (AAA AAA AAG CTT
TCA GGC CAG CGT GTG CAT CCA CC). The gene fragment
cdaPS3-PCP10 encoding the PCP of module 10 (fragment of SCO3232,
cdaPS3) was amplified using the oligonucleotides 5′-cdaPS3-PCP10
(AAA AAA GGA TCC ACC GGC CGG ACC GCG GGC CG) and
8,19
1
8,19
the biosynthesis of the 3-MeGlu-containing compounds.
These results show that GlmT, DptI, and presumably also LptI
are methyltransferases involved in the biosynthesis of 3-MeGlu
residues in CDA, daptomycin, and A54145. However, the
substrate and mechanism of the methylation reaction and the
nature of the other possible steps in the biosynthetic pathway
remained unclear.
3
′-cdaPS3-PCP10 (AAA AAA GCGG CCGC GCC CTT CGC CCC
GGC GAG CAC). The gene dptI was amplified using the primer
combination of 5′-dptI (AAA AAA CCA TGG TGA CCG GCG AAA
CCC GCA CCA C) and 3′-dptI (AAA AAA AAG CTT TGG TTT
GCG TCC GTG GGC GAC GA). For amplification of lptI, the
oligonucleotides 5′-lptI (AAA AAA GGA TCC ATG CAG GCG GAT
GCA CCG GCG G) and 3′-lptI (AAA AAA AAG CTT TCA GGT
GGG TGG CTT GTG GGA GAC GG) were used.
After purification and digestion with NcoI, HindIII (glmT, dptI),
BamHI, HindIII (ilVE, lptI), and NcoI, NotI (cdaPS3-PCP10), respec-
tively, the gene fragments of glmT, ilVE, dptI, and lptI were ligated
into the corresponding restriction sites of a derivatized pET-28(a+)
vector (Novagen), whereas the gene fragment cdaPS3-PCP10 was
similarly ligated into a derivatized pQE30 vector (Qiagen).
(
17) Kempter, C.; Kaiser, D.; Haag, S.; Nicholson, G.; Gnau, V.; Walk, T.;
Gierling, K. H.; Decker, H., Z a¨ hner, H.; Jung, G.; Metzger, J. W. Angew.
Chem., Int. Ed. Engl. 1997, 36, 498-501.
(
(
(
18) Nguyen, K. T.; Kau, D.; Gu, J. Q.; Brian, P.; Wrigley, S. K.; Baltz, R. H.;
Miao, V. Mol. Microbiol. 2006, 61, 1294-1307.
19) Milne, C.; Powell, A.; Jim, J.; Al, Nakeeb, M.; Smith, C. P.; Micklefield,
J. J. Am. Chem. Soc. 2006, 128, 11250-11259.
20) Debono, M.; Barnhart, M.; Carrell, C. B.; Hoffmann, J. A.; Occolowitz, J.
L.; Abbott, B. J.; Fukuda, D. S.; Hamill, R. L.; Biemann, K.; Herlihy, W.
C. J. Antibiot. 1987, 40, 761-777.
(
(
21) Kagan, H. M.; Meister, A. Biochemistry 1966, 5, 725-732.
22) Gr u¨ newald, J.; Sieber, S. A.; Mahlert, C.; Linne, U.; Marahiel, M. A. J.
Am. Chem. Soc. 2004, 126, 17025-17031.
Production of Recombinant Enzymes. The pQE30- and pET-28-
(a+)-plasmids, containing the gene fragments of interest, were used to
transform E. coli BL21(DE3) (Novagen). For production of recombinant
(
(
(
23) Hartzoulakis, B.; Gani, D. J. Chem. Soc., Perkin Trans. 1994, 1, 2525-
2
531.
24) Soloshonok, V. A.; Cai, C.; Hruby, V. J.; Meervelt, L. V.; Mischenko, N.
Tetrahedron 1999, 55, 12031-12044.
25) Wehbe, J.; Rolland, V.; Roumestant, M. L.; Martinez, J. Tetrahedron:
Asymmetry 2003, 14, 1123-1126.
(26) Magarvey, N. A.; Haltli, B.; He, M.; Greenstein, M.; Hucul, J. A.
Antimicrob. Agents Chemother. 2006, 50, 2167-2177.
J. AM. CHEM. SOC.
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VOL. 129, NO. 39, 2007 12013