Vitamin B<sub>6</sub> Biosynthesis: Charting the Mechanistic Landscape

Fitzpatrick, Thérésa Bridget; Moccand, Cyril; Roux, Cécile

doi:10.1002/cbic.201000084

Cited by 20 publications

(13 citation statements)

References 54 publications

(109 reference statements)

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“…post chromophoric intermediate formation, and in particular the effect on G3P binding, we decided to probe product formation further. Two points are important to note in this context; firstly, the major rate-limiting step along the PLP biosynthesis reaction co-ordinate is in fact dissociation of the product [27] and secondly, the maximum absorbance for the formation of the product is at 414 nm in different buffers examined (i.e. phosphate, HEPES) at pH 8.0.…”

Section: Resultsmentioning

confidence: 99%

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It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

2011

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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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Section: Resultsmentioning

confidence: 99%

“…We next attempted to explain the observation that the final reaction product PLP is tightly bound to the enzyme [27], [31] in a more physiological context. As stated above, this represents a rate-limiting step in the overall biosynthesis reaction.…”

Section: Resultsmentioning

confidence: 99%

It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

2011

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“…There are three known enzymes in living systems that catalyze the production of PLP: PNP oxidase [3], [29], present in both prokaryotic and eukaryotic organisms; PL kinase, which is also widely distributed in nature [3], [11]; and PLP synthase, which is found in plants and many microorganisms [30]. Both PNP oxidase and PLP synthase have been shown to bind PLP tightly and to transfer the tightly bound PLP to an apo-B 6 enzyme [28], [31].…”

Section: Discussionmentioning

confidence: 99%

Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

et al. 2012

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Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B6 requiring enzymes. PLP reacts with apo-B6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.

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“…While the biosynthesis, interconversion and salvage pathways for vitamin B 6 species are well characterized (Fitzpatrick et al, 2010;Mooney & Hellmann, 2010;Herrero et al, 2011;Mukherjee et al, 2011;Sang et al, 2011;Rueschhoff et al, 2013;Szydlowski et al, 2013), little is known about the regulation of PLP synthesis or about the connection between PLP and general metabolism (Shi et al, 2002;Chen & Xiong, 2005;Titiz et al, 2006;Rueschhoff et al, 2013;Vanderschuren et al, 2013). How PLP molecules are delivered to their target enzymes 3These authors contributed equally to this work.…”

Section: Introductionmentioning

confidence: 99%

Evidence that COG0325 proteins are involved in PLP homeostasis

et al. 2016

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Pyridoxal 59-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 59-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.

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Vitamin B₆ Biosynthesis: Charting the Mechanistic Landscape

Abstract: This paper is dedicated to Nikolas Amrhein (ETH Zürich) for his outstanding support over the years together in Zürich unravelling the alternative route of vitamin B 6 biosynthesis in micro-organisms and plants.

Cited by 20 publications

References 54 publications

It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Evidence that COG0325 proteins are involved in PLP homeostasis

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Vitamin B6 Biosynthesis: Charting the Mechanistic Landscape

Abstract: This paper is dedicated to Nikolas Amrhein (ETH Zürich) for his outstanding support over the years together in Zürich unravelling the alternative route of vitamin B 6 biosynthesis in micro-organisms and plants.

Cited by 20 publications

References 54 publications

It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Evidence that COG0325 proteins are involved in PLP homeostasis

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Vitamin B₆ Biosynthesis: Charting the Mechanistic Landscape