Understanding the role of pyridoxal reductase (PDXI) in pyridoxal 5’-phosphate (PLP) homeostasis.

Presented During:

07/31/2022: 5:30 PM - 7:30 PM
Portland Marriott Downtown Waterfront  


2022: 72nd ACA Annual Meeting

Session Type:


Presenting Author :

Akua Donkor  
Virginia Commonwealth University

Additional Author(s):

Mohini Ghatge  
Virginia Commonwealth University
Faik Musayev  
Virginia Commonwealth University
Roberto Contestabile  
Sapienza University of Rome
Martino Di Salvo  
Sapienza University of Rome
Martin Safo  
Virginia commonwealth university

Abstract Body:

Purpose: Pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6 is a cofactor for over 180 B6 enzymes (PLP-dependent enzymes) that are involved in critical biochemical reactions, e.g. amino acid, heme, and neurotransmitter biosynthesis. In prokaryotes, yeasts and plants, PLP is obtained from de novo and salvage pathways. The salvage pathway involves the enzymes pyridoxal kinase (PL kinase) and pyridoxine 5'-phosphate oxidase (PNPO) that use the B6 vitamers, pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM) to produce PLP. Humans can only synthesize PLP through the salvage pathway. Deficiency of PLP in the cell leads to several diseases. PLP is a highly reactive molecule and toxic, therefore its concentration in the cell is regulated by phosphatases that dephosphorylate it to PL and/or feedback inhibition of PL kinase and PNPO.
A third salvage enzyme, PDXI, with a reductase activity was recently discovered in bacteria and plants, catalyzing PL in the presence of NADPH to form PN and NADP+. Despite, its importance as a PLP homeostasis protein for efficient salvage of PL, only limited information on its structure and function is known. This study is aimed at characterizing E. coli PDXI with respect to its catalytic conversion of PL to PN, substrate binding specificity, regulation, and atomic structure.
Method: E. coli cells containing the cloned pdxI were grown in 6 liters of LB media and after reaching an O.D.600nm of greater than 0.6, were induced with 0.5mM IPTG. Cells were grown for an additional 4 hours at 37 C and harvested by centrifugation. The expressed PDXI was purified using a nickel column via fast protein liquid chromatography (FPLC). Protein fractions with single band at 33 kDa as checked by SDS-PAGE were combined and dialyzed overnight in a buffer containing 50mM NaH2PO4, 150mM NaCl, pH 7.5. The pure apo-protein (>90%) as judged by SDS-PAGE was used for crystallization with the Crystal Gryphon robot using a wide range of crystallization conditions. The condition 0.1 M MgCl, 0.1 M MES:NaOH, pH 6.5, and 30% PEG 400 gave the best crystal, which was used to collect X-ray diffraction data. The crystal structure of PDXI has been solved by a molecular replacement with the Phenix program, and the model subsequently refined using the Phenix and COOT programs. Further crystallographic studies of PDXI in complex with its substrates PL and/or NADPH or its products PN and/or NADP+ are ongoing. Kinetic studies are also ongoing using UV/vis spectroscopic method by measuring a decrease of NADPH at 340 nm. The mechanism and kinetic constants (km and Kcat) for PDXI will be determined. ITC and/or MST measurements will also be used to analyze the binding (Kd) of B6 vitamers to apo-PDXI to study substrate specificity.
Results: The crystal structure of apo-PDXI has been determined to 2.2 Å. This is the first such structure of a PDXI protein. PDXI folds as a TIM barrel and consists of 11 α-helices and 8 β-strands. Specific interactions of PL or NADPH with the protein await further crystallographic study. The kinetic and binding studies are also ongoing.
Conclusion: It is expected that the study will provide detailed molecular level information that will be useful in understanding PDXI cellular function and provide insight into PLP homeostasis.

Additional Information - Poster (2022)

Are you registered for the ACA meeting?


For scheduling purposes please select a session that most matches your poster topic. If your poster theme does not align with the topics of the sessions, please select a general category.

2.1.5 What I Learned from My First Structures

If submitted for a poster would you like to be considered for a poster prize?


If yes, which poster prize?

RCSB Protein Data Bank