Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase

doi:10.1073/pnas.0610007104

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase

*Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027;
^†School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, United Kingdom; and
^‡Center for Advanced Biotechnology and Medicine and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, NJ 08854

Communicated by Wayne A. Hendrickson, Columbia University, New York, NY, November 13, 2006 (received for review September 25, 2006)

Abstract

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate l-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the l-stereospecificity. A second, possibly allosteric, l-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of l-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

Footnotes

^§To whom correspondence should be addressed. E-mail: ltong{at}columbia.edu
Author contributions: F.F., J.L.R.A., and C.G.M. contributed equally to this work; F.F., J.L.R.A., C.G.M., G.T.M., S.K.C., and L.T. designed research; F.F., J.L.R.A., C.G.M., S.M.V., A.H., M.A., C.B., S.J.T., J.S., T.T., R.X., L.-C.M., L.Z., and T.B.A. performed research; F.F., J.L.R.A., C.G.M., S.M.V., A.H., M.A., C.B., S.J.T., J.S., R.X., L.-C.M., T.B.A., G.T.M., S.K.C., and L.T. analyzed data; and F.F., J.L.R.A., C.G.M., T.B.A., G.T.M., S.K.C., and L.T. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 1YW0, 2NW7, 2NW8, 2NW9, 1ZEE, and 2NWB).
This article contains supporting information online at www.pnas.org/cgi/content/full/0610007104/DC1.
Abbreviations:

d-Trp,

d-tryptophan;

IDO,

indoleamine 2,3-dioxygenase;

l-Trp,

l-tryptophan;

TDO,

tryptophan 2,3-dioxygenase.