RESEARCH LETTER
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through a single pericyclic transition state such as 6. Therefore, a
stepwise [412] cycloaddition mechanism, for example, one involving
a dipolar intermediate such as 7, cannot at present be ruled out. In
contrast, the C-C bond formation catalysed by SpnL may involve a
Rauhut–Currier mechanism15 consistent with the observation that 10
is susceptible towards nucleophilic addition by a thiol (see Supplemen-
tary Information Section 3.10) forming a covalent adduct that may be
structurally analogous to 11 or 12 (see Fig. 4), although the specific site
of attack remains unknown. Whereas these mechanistic proposals are
at present speculative, it is worth noting that Roush and co-workers
were able to accomplish their non-enzymatic total synthesis of
spinosyn A by exploiting both the transannular Diels–Alder and
Rauhut–Currier reactions in an analogous fashion25. This precedent
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mechanisms proposed for the SpnF- and SpnL-catalysed reactions.
In summary, the biosynthetic pathway for spinosyn A is now fully
established (Fig. 4), with the specific functions of SpnM as a dehydra-
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steps biochemically determined. SpnF represents the first enzyme for
which specific acceleration of a [412] cycloaddition reaction has been
experimentally verifiedas itsonly knownfunction. Itstandsin contrast
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thesis7,8, and hydride transfer12, respectively, in addition to the [412]
cycloaddition reactions, the concertedness of which have yet to be
verified. For this reason, the SpnF reaction provides a unique system
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METHODS SUMMARY
All proteins used in thiswork were overexpressed in E.coliBL21(DE3)*(Invitrogen)
and purified by Ni-NTA (Qiagen) affinity chromatography. Specifically, SpnF was
co-overexpressed with the chaperone protein pair, GroEL/ES, to improve its
solubility. Because overexpression of the protein encoded by the originally
assigned spnM gene16 failed to afford an active soluble protein, the gene sequence
was re-examined and revised to include 204 additional base pairs (Supplementary
Fig. 2). Overexpression of the revised spnM gene produced an active enzyme with
significantly improved protein yield. All enzyme reaction products (5, 8, 10 and 13)
were extracted with ethyl acetate or chloroform and purified using silica gel column
chromatography or HPLC. Their structures were characterized by 1D- and 2D-
NMR spectroscopy and/or high-resolution mass spectrometry. In particular, the
stereochemistry of 8 was assigned based on its 1H-1H nuclear Overhauser enhance-
ment spectroscopy (NOESY) spectrum. All substrate specificity and time-course
assays were run in 50mM Tris-HCl buffer (pH 8) at 30uC. Reaction aliquots were
quenched with an excess volume of ethanol after a given incubation time and
centrifuged to remove protein. The supernatant was then analysed by reverse phase
HPLCwithdetectionbyultravioletabsorbanceat254 nm(Fig. 2)or280 nm(Fig. 3).
Time course assays also included p-methoxyacetophenone as an internal standard
to normalize the peak areas corresponding to 5. Numerical integration of equations
(1) and (2) used the fourth order Runge–Kutta algorithm28 following non-dimen-
sionalization of substrate concentration. The resulting simulated progress curves
were fitusingsteepestdescent29 directly tofulltime-coursesof normalizedsubstrate
concentration obtained via the HPLC discontinuous assay to provide the kinetic
parameters and a concentration normalization factor. Further detail regarding
experimental procedures as well as data fitting and analysis is described in the
Supplementary Information.
˜
´
26. Guimaraes, C. R. W., Udier-Blagovic, M. & Jørgensen, W. L. Macrophomate
synthase: QM/MM simulations address the Diels–Alder versus Michael–aldol
reaction mechanism. J. Am. Chem. Soc. 127, 3577–3588 (2005).
27. Serafimov, J. M., Gillingham, D., Kuster, S. & Hilvert, D. The putative Diels–Alderase
macrophomate synthase is an efficient aldolase. J. Am. Chem. Soc. 130,
7798–7799 (2008).
28. Tenenbaum, M. & Pollard, H. Ordinary Differential Equations (Dover, 1985).
29. Draper, N. R. & Smith, H. Applied Regression Analysis 3rd edn (Wiley-Interscience,
1998).
Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank C. Whitman for review of the manuscript, L. Hong for
carrying out the early cloning work, B. Shoulders and S. Sorey for assistance with the
interpretation of the NMR spectra, and S. Mansoorabadi and E. Isiorho for discussions
on the reaction mechanisms and structural modelling of SpnF. This work is supported
in part by grants from theNationalInstitutes of Health (GM035906, F32AI082906), the
Texas Higher Education Coordination Board (ARP-003658-0093-2007), and the
Welch Foundation (F-1511).
Author Contributions H.-w.L. provided the scientific direction and the overall
experimental design for the studies. H.J.K. designed and performed most of the
experiments. S.-h.C. participated in the chemical synthesis of the substrate for SpnJ (2)
and the characterization of the structures of the enzyme reaction products. M.W.R.
analysed the kinetic experiment data. Y.-n.L. carried out the mutation studies of SpnF.
H.J.K., M.W.R. and H.-w.L. wrote the manuscript.
Received 29 March 2010; accepted 3 March 2011.
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