COMUNICAZIONE ORALE A CONGRESSO SU INVITO “On the mechanism of addition of pyridoxal 5’-phosphate to serine hydroxymethyltransferase”, in The Third International Conference on Cofactors (ICC03), Finland, Turku, 10-15 July 2011.

ON THE MECHANISM OF PYRIDOXAL 5’-PHOSPHATE ADDITION TO SERINE HYDROXYMETHYLTRANSFERASE. Roberto Contestabile1, Rita Florio1, Isabel Noguès2, Martin K. Safo3, Verne Schirch3 and Martino Luigi di Salvo1 1Dipartimento di Scienze Biochimiche and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Italy, w3.uniroma1.it/bio_chem/sito_biochimica/EN/index.html 2Istituto di Biologia Agroambientale e Forestale, Centro Nazionale delle Ricerche, Monterotondo Scalo, Roma, Italy. 3Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA, USA. The addition of cofactors to newly synthesized apo-proteins in the cell is a crucial event that can occur at different stages of polypeptide folding and affect it to a variable extent. The form in which the cofactor is available in the intracellular medium may also differ according to its reactivity or toxicity. Although much work has been done on the mechanism and structure of vitamin B6–dependent enzymes, little is known on how pyridoxal 5’phosphate (PLP) is supplied to the apoenzymes in order to meet their requirement in terms of cofactor. Free PLP availability in the cell is significantly limited by the high reactivity of its aldehyde group, forming aldimines with amino groups on non-B6 enzymes and amino acids, as well as by dephosphorylation by phosphatases. This raises the intriguing question of how the cell supplies sufficient PLP, with high specificity, to the dozens of B6 enzymes. The traditionally proposed mechanism involves a release of PLP from the enzymes that catalyze its formation into solution, where it finds its way to the active site of apo-B6 enzymes. An obvious problem with this mode of PLP addition is the potential interactions of the cofactor with nucleophiles and its dephosphorylation by phosphatases, which would significantly deplete the free PLP availability. An alternative mechanism for PLP addition involves a direct channeling from the enzymes that produce it in the cell to the acceptor B6 enzymes, thus avoiding its release into solution. This mechanism offers an efficient, exclusive, and protected means of delivery of the reactive PLP. As we approached this matter, we chose Escherichia coli as a model system. To start with, we decided to use serine hydroxymethyltransferase (eSHMT) as PLP acceptor, since this is a well characterized fold type I B6 enzyme. A first step was the understanding of how free PLP reacts in vitro with apo-eSHMT to form the active holoenzyme. Thereafter, we focused on the mechanism of PLP transfer from the enzymes that catalyse its formation in the E. coli cell, pyridoxine phosphate oxidase and pyridoxal kinase, to eSHMT. The transfer kinetic studies were performed by means of UV-visible spectroscopy, circular dichroism measurements and chemical quenched-flow experiments. Our presentation will The results obtained are in favor of a channeling mechanism.