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It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.
Type of publication
Peer-reviewed
Publikationsform
Original article (peer-reviewed)
Author
Moccand Cyril, Kaufmann Markus, Fitzpatrick Teresa B,
Project
Defining vitamin B1 and B6 metabolism in plants: synthesis, regulation and transport
Show all
Original article (peer-reviewed)
Journal
PloS one
Volume (Issue)
6(1)
Page(s)
16042 - 16042
Title of proceedings
PloS one
DOI
10.1371/journal.pone.0016042
Open Access
URL
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016042
Type of Open Access
Website
Abstract
The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2) that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.
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