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

Abstract

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.

Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii

“…Protein subunit concentration was calculated using a theoretical extinction coefficient at 280 nm of 66 700 M À1 Ácm À1 (calculated with the Expasy PROTPARAM tool; http://web.expasy.org/protparam). PLP content was determined as described previously [49,50]. Apo-PdxR was prepared using L-cysteine as previously described for E. coli SHMT [51] except that extensive dialysis was used instead of phenyl-sepharose chromatography to remove thiazolidine complex formed between PLP and cysteine.…”

Identification of YbhA as the pyridoxal 5ʹ-phosphate (PLP) phosphatase in <i>Escherichia coli</i>: Importance of PLP homeostasis on the bacterial growth

“…To maintain the PLP homeostasis, the final enzymes involved in PLP biosynthesis, PdxK and PdxH, are directly inhibited by PLP in E. coli (Ghatge et al, 2012;Zhao and Winkler, 1995). In addition, PLP phosphatase, which dephosphorylates PLP and decreases the PLP concentration, has been considered to be another mechanism that maintains PLP homeostasis.…”

“…The white arrow indicates the dephosphorylation of PLP into PL, which is proposed in this study. PdxH and PdxK are subjected to feedback inhibition by PLP (Ghatge et al, 2012;Zhao and Winkler, 1995). Abbreviations: G3P; glycerol 3-phosphate, PL; pyridoxal, PM; pyridoxamine, PN; pyridoxine, PMP; pyridoxamine 5¢-phosphate, PNP; pyridoxine 5¢-phosphate, PLP; pyridoxal 5¢-phosphate.…”

“…However, the conversion of PM or PN to PLP requires two steps: the sequential phosphorylation and oxidation by PdxK and PdxH (PNP/PMP oxidase). The feedback regulation of PdxK and PdxH by PLP occurs and may explain why the addition of PM or PN has no effect (Ghatge et al, 2012;Zhao and Winkler, 1995). Therefore, in this study, we used PL as a substrate to induce an excess PLP condition.…”

“…The feedback regulation also includes PLP-dependent inhibition of PdxK (PL/PM/PN kinase) which is involved in the salvage pathway ( Fig. 1) (Ghatge et al, 2012;Roseann and Walter, 1970;Zhao and Winkler, 1995). In addition, the PLP phosphatase that counteracts the PL kinase may be a potential mechanism in homeostasis.…”