C O M M U N I C A T I O N S
Archaeal genomes clearly are a rich source of proteins; however,
genome annotation based on primary sequence homologies some-
times does not even hint at the true synthetic potential of the
corresponding proteins. Chemical function analysis is required,
ideally, with a range of substrates to ascertain the most likely in
vivo function and to uncover unexpected properties. The archaeal
gene denoted originally as a glucose-1-phosphate deoxythymidy-
lyltransferase is actually a bifunctional acetyltransferase/N-acetyl-
glucosamine-1-phosphate uridylyltransferase. The use of thermo-
stable enzymes in synthetic schemes also allows chemical steps
that are sluggish at lower temperatures to be incorporated into the
same step with an enzyme. Current studies are underway to use
these tagged sugar nucleotides to probe protein modifications by
N-acetylglucosamine moieties.
Acknowledgment. We are grateful for an NSF-CAREER award
and a Shimadzu University Research Grant, and we thank the
Herman Frasch Foundation, administered by the American Chemi-
cal Society, for support of this research. N.L.P. is a Cottrell Scholar
of Research Corporation.
Figure 2. Acyltransferase activity of the bifunctional enzyme with natural
and truncated acetylCoA derivatives. R ) CH3, CH2Cl, CCH.
in the analogous bacterial enzyme,10 the enzyme tolerated a propyl
group reasonably well with only an 8-fold decrease in kcat/KM, but
a two-carbon extension proved detrimental to efficient product
formation.
Supporting Information Available: Experimental details for
cloning, expression, and purification of the hyperthermophilic enzyme,
details of the mass spectrometry assays including calibration curves,
and synthetic details for thioesters 9 and 10. This material is available
Reexamination of the protein sequence indeed shows alignment
of the first 240 amino acids with NTP-transferases. However, the
C-terminal portion contains a hexapeptide repeat motif often
associated with acyltransferase activity.11 Analysis of a structure
of the analogous bifunctional enzyme from Escherichia coli with
bound acetyl CoA (7) shows several contacts between CoA and
the protein. However, no contacts are made with the acyl side chain,
and the diphosphate is solvent exposed.12 Fatty acid synthases
(FASs) and polyketide synthases (PKSs) are known to accept
truncated CoA thioesters as substrates, but these enzymes form
covalent acylated intermediates before transfer to a bound sub-
strate.13 The structure of the bifunctional E. coli enzyme does not
support a covalent enzyme intermediate for the acyltransfer reaction
to glucosamine.
References
(1) (a) Tai, H.-C.; Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson,
L. C. J. Am. Chem. Soc. 2004, 126, 10500-105001. (b) Vocadlo, D. J.;
Hang, H. C.; Kim, E.-J.; Hanover, J. A.; Bertozzi, C. R. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 9116-9121. (c) Zachara, N. E.; Hart, G. W. Chem.
ReV. 2002, 102, 431-438.
(2) (a) Khidekel, N.; Arndt, S.; Lamarre-Vincent, N.; Lippert, A.; Poulin-
Kerstein, K. G.; Ramakrisnan, B.; Qasba, P. K.; Hsieh-Wilson, L. C. J.
Am. Chem. Soc. 2003, 125, 16162-16163. (b) Haynes, P. A.; Aebersold,
R. Anal. Chem. 2002, 72, 5402-5410. (c) Wells, L.; Vosseller, K.; Cole,
R. N.; Cronshaw, J. M.; Matunis, M. J.; Hart, G. W. Mol. Cell. Proteomics
2002, 1, 791-804.
(3) Thorson, J. S.; Barton, W. A.; Hoffmeister, D.; Albermann, C.; Nikolov,
D. B. ChemBioChem 2004, 5, 16-25. (b) Koeller, K. M.; Wong, C.-H.
Nat. Biotechnol. 2000, 18, 835-841. (c) Elhalabi, K.; Rice, K. G. Curr.
Med. Chem. 1999, 6, 93-116.
The ability of FASs and PKSs to accept significantly cheaper
and less synthetically challenging analogues such as the N-
acetylcysteamine thioester 8 has greatly aided experiments to
understand and exploit these enzymes; therefore, we decided to
synthesize and test 8 with the P. furiosus enzyme. In fact, this
truncated analogue was readily accepted and turned over with only
a 2-3-fold reduction in kcat/KM compared to the full length substrate
(Table 1). Emboldened by this discovery, we synthesized analogues
9 and 10 (Figure 2) containing sterically conservative modifications
that are nevertheless amenable to selective tagging reactions via
thiol substitution14 or Sonogashira reactions15 and alkyne/azide
coupling,16 respectively. The enzyme not only was able to acylate
the amine but also could convert the resulting modified glucos-
amine-1-phosphate into the activated UDP-sugar with 80% conver-
sion in one pot. The acylation reactions did not occur at 37 °C
with enzyme or at 80 °C without enzyme with one exception.
Significant uncatalyzed acylation took place with the alkyne
analogue at 80 °C. Therefore, even if the acyltransfer domain did
not readily accept this modification, the thermostability of the
enzyme allows the coupling of the chemical acylation with the
enzymatic uridyl transfer in one pot. For comparison, the chemical
synthesis of an azide-labeled UDP-GlcNAc required 11 chemical
steps with less than a 15% overall yield.1b
(4) Rivera, M. C.; Lake, J. A. Nature 2004, 431, 152-155.
(5) Sterner, R.; Liebl, W. Crit. ReV. Biochem. Mol. Biol. 2001, 36, 39-106.
(6) Adams, M. W. W.; Dailey, H. A.; DeLucas, L. J.; Luo, M.; Prestegaard,
J. H.; Rose, J. P.; Wang, B.-C. Acc. Chem. Res. 2003, 36, 191-198.
(7) Zea, C. J.; Pohl, N. L. Anal. Biochem. 2004, 328, 196-202.
(8) Mengin-Lecreulx, D.; Heijenoort, J. V. J. Bacteriol. 1994, 18, 5788-
5795.
(9) Riddles, P. W.; Blakeley, R. L.; Zenner, B. Methods Enzymol. 1983, 91,
49-60.
(10) Gehring, A. M.; Lees, W. J.; Mindiola, D. J.; Walsh, C. T.; Brown, E. D.
Biochemistry 1996, 35, 579-585.
(11) (a) Bairoch, A. Nucleic Acids Res. 1993, 21, 3097-3103. (b) Dicker, I.
B.; Seetharam, S. Mol. Microbiol. 1992, 6, 817-823. (c) Vourio, R.;
Harkonen, T.; Tolvanen, M.; Vaara, M. FEBS Lett. 1994, 337, 289-292.
(12) Olsen, L. R.; Roderick, S. L. Biochemistry 2001, 40, 1913-1921.
(13) See, for example: (a) Mishra, P. K.; Drueckhammer, D. G. Chem. ReV.
2000, 100, 3283-3309. (b) Pohl, N. L.; Gokhale, R. S.; Cane, D. E.;
Khosla, C. J. Am. Chem. Soc. 1998, 120, 11206-11207.
(14) Svensson, R.; Greno¨, C.; Johansson, A.-S.; Mannervik, B.; Morgenstern,
R. Anal. Biochem. 2002, 311, 171-178.
(15) (a) Bong, D. T.; Ghadiri, M. R. Org. Lett. 2000, 3, 2509-2511. (b)
Dibowski, H.; Schmidtchen, F. P. Angew. Chem., Int. Ed. 1998, 37, 476-
478.
(16) Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535-546. (b) Lewis,
W. G.; Magallon, F. G.; Fokin, V. V.; Finn, M. G. J. Am. Chem. Soc.
2004, 126, 9152-9153. (c) Link, J. A.; Vink, M. K. S.; Tirrell, D. A. J.
Am. Chem. Soc. 2004, 126, 10598-10602. (d) Speers, A. E.; Adam, G.
C.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686-4687. (e)
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596-2599. (f) Huisgen, R. Pure Appl. Chem.
1989, 61, 613-628.
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