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J. Biol. Chem., Vol. 282, Issue 9, 6098-6105, March 2, 2007

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Reaction Mechanism of Pyridoxal 5'-Phosphate Synthase

DETECTION OF AN ENZYME-BOUND CHROMOPHORIC INTERMEDIATE^*

Thomas Raschle, Duilio Arigoni, René Brunisholz^¶, Helene Rechsteiner^¶, Nikolaus Amrhein, and Teresa B. Fitzpatrick¹

From the Institute of Plant Sciences, ETH Zurich, 8092 Zurich, the Organic Chemistry Laboratory, ETH Zurich, 8093 Zurich, and the ^¶Functional Genomics Center Zurich, 8057 Zurich, Switzerland

Vitamin B6 is an essential metabolite in all organisms. De novo synthesis of the vitamin can occur through either of two mutually exclusive pathways referred to as deoxyxylulose 5-phosphate-dependent and deoxyxylulose 5-phosphate-independent. The latter pathway has only recently been discovered and is distinguished by the presence of two genes, Pdx1 and Pdx2, encoding the synthase and glutaminase subunit of PLP synthase, respectively. In the presence of ammonia, the synthase alone displays an exceptional polymorphic synthetic ability in carrying out a complex set of reactions, including pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, that convert C3 and C5 precursors into the cofactor B6 vitamer, pyridoxal 5'-phosphate. Here, employing the Bacillus subtilis proteins, we demonstrate key features along the catalytic path. We show that ribose 5-phosphate is the preferred C5 substrate and provide unequivocal evidence that the pent(ul)ose phosphate imine occurs at lysine 81 rather than lysine 149 as previously postulated. While this study was under review, corroborative crystallographic evidence has been provided for imine formation with the corresponding lysine group in the enzyme from Thermotoga maritima (Zein, F., Zhang, Y., Kang, Y.-N., Burns, K., Begley, T. P., and Ealick, S. E. (2006) Biochemistry 45, 14609–14620). We have detected an unanticipated covalent reaction intermediate that occurs subsequent to imine formation and is dependent on the presence of Pdx2 and glutamine. This step most likely primes the enzyme for acceptance of the triose sugar, ultimately leading to formation of the pyridine ring. Two alternative structures are proposed for the chromophoric intermediate, both of which require substantial modifications of the proposed mechanism.

Received for publication, November 15, 2006 , and in revised form, December 20, 2006.

Addendum—In a publication that appeared after the original submission of this work, independent x-ray crystallographic evidence has been presented for imine formation at the equivalent lysine (lysine 82) in the enzyme from Thermotoga maritima (1). While this study was under review, the crystal structure of Pdx1 from T. maritima with ribulose 5-phosphate bound was published, where imine formation with the pentulose phosphate was observed at Lys-82 (equivalent to Lys-81 in BsPdx1) (1).

^* This work was supported by Swiss National Science Foundation Grant 3100A0-107975/1 (to N. A. and T. B. F.) and by a grant from Novartis International AG, Basel (to D. A.). 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.

¹ To whom correspondence should be addressed. Tel.: 41-44-6323841; Fax: 41-44-6321044; E-mail: tfitzpatrick{at}ethz.ch.

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