Figure 1. Formation of (A) chalcone by CHS, (B) SEK4/SEK4b by OKS (and CHS S338V), and (C) other polyketide products.
4
Streptomyces coelicolor and the longest polyketides gener-
previous papers, we reported that the residue 197 determines
the polyketide chain length and product specificities in the
octaketide-producing A. arborescens OKS and the penta-
3
ated by the structurally simple type III PKS. In A. arbore-
scens OKS, the CHS’s active-site residues Thr197, Gly256,
and Ser338 (numbering in Medicago satiVa CHS ) are
2
a
3
ketide-producing A. arborescens PCS.
uniquely replaced with Gly, Leu, and Val, respectively
(
To further study the structure-function relationship be-
T197G/G256L/S338V).3b Interestingly, the three residues
tween CHS and OKS enzyme, here we constructed a series
7
lining the active-site cavity are sterically altered in a number
of functionally divergent type III PKSs including A. arbore-
scens pentaketide chromone synthase (PCS) (T197M/G256L/
of Scutellaria baicalensis CHS mutants in which the three
residues were changed from those in CHS to those in OKS
(T197G, G256L, and S338V), and investigated the mecha-
nistic consequences of the mutations using 4-coumaloyl-CoA
and/or malonyl-CoA as substrates.
Interestingly, in the absence of the coumaroyl starter, both
wild-type and the mutant S. baicalensis CHSs initiated
decarboxylative condensation of malonyl-CoA, but most of
the polyketide chain elongation reactions were terminated
at the triketide stage to predominantly produce triacetic acid
lactone (TAL) (Figure 2A). This is in good agreement with
an earlier report that mutation of M. satiVa CHS at the
residues (T197L, G256L, and S338I) resulted in functional
3a
S338V) , Rheum palmatum aloesone synthase (T197A/
5
G256L/S338T), and Gerbera hybrida 2-pyrone synthase
6
(
2PS) (T197L/G256L/S338I). These chemically inert resi-
dues have been shown to control starter substrate and product
specificity by steric modulation of the active-site cavity in
2d,6b
M. satiVa CHS and in G. hybrida 2PS.
Further, in
(3) (a) Abe, I.; Utsumi, Y,; Oguro, S.; Morita, H.; Sano, Y.; Noguchi,
H. J. Am. Chem. Soc. 2005, 127, 1362-1363. (b) Abe, I.; Oguro, S.; Utsumi,
Y,; Sano, Y.; Noguchi, H. J. Am. Chem. Soc. 2005, 127, 12709-12716.
(4) (a) Fu, H.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. J. Am.
Chem. Soc. 1994, 116, 4166-4170. (b) Fu, H.; Hopwood, D. A.; Khosla,
C. Chem. Biol. 1994, 1, 205-210.
(
5) (a) Abe, I.; Utsumi, Y,; Oguro, S.; Noguchi, H. FEBS Lett. 2004,
(7) (a) Abe, I.; Morita, H.; Nomura, A.; Noguchi, H. J. Am. Chem. Soc.
2000, 122, 11242-11243. (b) The deduced amino acid sequences of S.
baicalensis CHS showed 77.4% (302/389) identity with those of M. satiVa
CHS and 60.4% (235/389) identity with A. arboresens OKS. The
recombinant enzyme with an additional hexahistidine tag at the C-terminal
was expressed in E. coli and purified by Ni-chelate chromatography as
5
2
62, 171-176. (b) Abe, I.; Watanabe, T.; Lou, W.; Noguchi, H. FEBS J.
006, 273, 208-218.
(6) (a) Eckermann, S.; Schr o¨ der, G.; Schmidt, J.; Strack, D.; Edrada, R.
A.; Helariutta, Y.; Elomaa, P.; Kotilainen, M.; Kilpel a¨ inen, I.; Proksch, P.;
Teeri, T. H.; Schr o¨ der, J. Nature 1998, 396, 387-390. (b) Jez, J. M.; Austin,
M. B.; Ferrer, J.; Bowman, M. E.; Schr o¨ der, J.; Noel, J. P. Chem. Biol.
7a
described before. The wild-type enzyme showed KM ) 36.1 µM and kcat
-
1
2
000, 7, 919-930.
) 1.26 min for 4-coumaroyl-CoA.
500
Org. Lett., Vol. 8, No. 3, 2006