title: Inactive mutants of human pyridoxine 5'-phosphate oxidase: a possible role for a noncatalytic pyridoxal 5'-phosphate tight binding site.
abstract: Pyridoxal 5'-phosphate (PLP) is a cofactor for many vitamin B6-requiring enzymes that are important for the synthesis of neurotransmitters. Pyridoxine 5'-phosphate oxidase (PNPO) is one of two enzymes that produce PLP. Some 16 known mutations in human PNPO (hPNPO), including R95C and R229W, lead to deficiency of PLP in the cell and have been shown to cause neonatal epileptic encephalopathy (NEE). This disorder has no effective treatment, and is often fatal unless treated with PLP. In this study, we show that R95C hPNPO exhibits a 15-fold reduction in affinity for the FMN cofactor, a 71-fold decrease in affinity for the substrate PNP, a 4.9-fold decrease in specific activity, and a 343-fold reduction in catalytic activity, compared to the wild-type enzyme. We have reported similar findings for R229W hPNPO. This report also shows that wild-type, R95C and R229W hPNPO bind PLP tightly at a noncatalytic site and transfer it to activate an apo-B6 enzyme into the catalytically active holo-form. We also show for the first time that hPNPO forms specific interactions with several B6 enzymes with dissociation constants ranging from 0.3 to 12.3 μm. Our results suggest a possible in vivo role for the tight binding of PLP in hPNPO, whether wild-type or variant, by protecting the very reactive PLP, and transferring this PLP directly to activate apo-B6 enzymes.
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Mohini S
authors.initials: MS
authors.lastname: Ghatge
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Sayali S
authors.initials: SS
authors.lastname: Karve
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Tanya M S
authors.initials: TM
authors.lastname: David
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Mostafa H
authors.initials: MH
authors.lastname: Ahmed
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Faik N
authors.initials: FN
authors.lastname: Musayev
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Kendra
authors.initials: K
authors.lastname: Cunningham
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Verne
authors.initials: V
authors.lastname: Schirch
authors.affiliation: Department of Medicinal Chemistry School of Pharmacy and Institute for Structural Biology, Drug Discovery and Development Virginia Commonwealth University Richmond VA USA.
authors.forename: Martin K
authors.initials: MK
authors.lastname: Safo
keywords: enzyme mutation; neonatal epileptic encephalopathy; neurotransmitters; pyridoxal 5′‐phosphate; pyridoxine 5′‐phosphate oxidase; vitamin B6