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J. Biol. Chem., Vol. 284, Issue 2, 897-904, January 9, 2009

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Structural Basis of Enzymatic (S)-Norcoclaurine Biosynthesis^*

Andrea Ilari, Stefano Franceschini, Alessandra Bonamore, Fabio Arenghi, Bruno Botta^¶, Alberto Macone, Alessandra Pasquo^||, Luca Bellucci^**, and Alberto Boffi¹

From the Dipartimento di Scienze Biochimiche and Istituto di Biologia e Patologia Molecolari, CNR, Sapienza University, Piazzale Aldo Moro 5, 00185 Roma, Italy, CPC Biotech s.r.l., via dei Mille 111, 80121, Napoli, Italy, the ^¶Dipartimento di Chimica e Tecnologia del Farmaco Sapienza University, Piazzale Aldo Moro 5, 00185, Roma, Italy, ^||ENEA Casaccia Research Centre, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale via Anguillarese 301, I-00100 Roma, Italy, and the ^**Istituto Tecnico Chimico Farmacologico, University of Siena, via Aldo Moro 2, 53110, Siena, Italy

The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1Å resolution. NCS shares no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-Å-long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.

Received for publication, May 15, 2008 , and in revised form, November 6, 2008.

^* This work was supported by Project FIRB 2003 from Ministero dell' Istruzione dell' Universita' e della Ricerca Scientifica (to A. B., B. B., and F. A.), local university grants (to A. B. and B. B.), and the European Community Research Infrastructure Action under the FP6 Structuring the European Research Area Programme (through the Integrated Infrastructure Initiative "Integrating Activity on Synchroton and Free Electron Laser Science, Contract R II 3-CT-2004-506008). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental materials (Text and Figures).

The atomic coordinates and structure factors (codes 2VNE and 2VQ5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence should be addressed: Dipartimento di Scienze Biochimiche, Sapienza University, P. le Aldo Moro 5, 00185 Roma, Italy. Fax: 39-06-4991-0990; E-mail: alberto.boffi{at}uniroma1.it.

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