Structural and Functional Features of Pyridoxal Phosphate-Binding Protein: Comparison
Please note this is a comparison between Version 3 by Asunción Contreras and Version 2 by Camila Xu.

The pyridoxal phosphate-binding protein (PLPBP) family (also termed ProsC/PROSC or COG0325 family) members are found in all kingdoms of life, exemplified by the proteins YBL036C (yeast), YggS (Gram-negative bacteria), YlmE (Gram-positive bacteria), PipY (cyanobacteria), PLPHP (humans) and HTH5 (rice). 

  • cyanobacteria
  • nitrogen regulation
  • COG0325
  • PLPHP
  • PLPBP

1. Introduction

Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, constitute an ecologically and biotechnologically important phylum, responsible for the evolution of the oxygenic atmosphere, being the main contributors to marine primary production [1]. Their photosynthetic lifestyle and ease of cultivation make them ideal production systems for several high-value compounds, including biofuels [2]. Despite important breakthroughs in the genetic analysis of cyanobacteria, there is still a remarkable proportion of genes of unknown function in this phylum, many of which are presumably relevant to the biology of cyanobacteria.
The cyanobacterium Synechococcus elongatus PCC7942 (hereafter S. elongatus), the only photosynthetic organism for which the contribution of each gene to fitness has been evaluated so far [3], is being used as a model system to address fundamental questions concerning the photosynthetic lifestyle. More recently, the S. elongatus genome has been used as the reference organism to create a database for the Cyanobacterial-Linked Genome [4], accessible through an interactive platform “https://dfgm.ua.es/es/cyanobacterial-genetics/dclg/index.htm (accesed on 1 August 2022)”.
In bacteria and plants, 2-oxoglutarate (2-OG), a key metabolic signal of the intracellular carbon-to-nitrogen balance, is sensed by the highly conserved and widely distributed signal transduction protein PII. PII regulates the activity of proteins involved in nitrogen metabolism by direct protein–protein interactions [5]. In S. elongatus PII interacts with a small (89 residues) protein called PipX (PII-interacting protein X), which was initially identified in yeast two-hybrid analyses [6,7].
PipX was also found in searches for proteins interacting with NtcA, the global transcriptional regulator involved in nitrogen assimilation in cyanobacteria [8]. PipX stabilizes the conformation of NtcA which is transcriptionally active and probably helps the local recruitment of RNA polymerase to NtcA-dependent promoters [9]. At low 2-OG concentrations corresponding to nitrogen-excess conditions, the sequestration of PipX by PII renders PipX unavailable for NtcA binding and activation, reducing the expression of NtcA-dependent gene targets [9,10,11,12,13]. Partner swapping by PipX is enabled by its N-terminal Tudor-like domain (TLD/KOW), which provides contacts for both NtcA and PII. Complex formation with PipX increases the affinity of PII for ADP [9], and, conversely, the interaction between PII and PipX is highly sensitive to fluctuations in the ATP/ADP ratio [14]. Thus, PipX partner swapping between PII and NtcA integrates signaling of the carbon-to-nitrogen ratio and the energy status by PII with the regulation of nitrogen-responsive genes controlled by NtcA [10,15,16].
Interestingly, a high PipX/PII ratio prevents growth [11,17] and, consistent with this, cyanobacterial genomes always contain at least as many copies of glnB as of pipX [18], suggesting that a relatively high ratio of PII over PipX is required to counteract unwanted interactions with low-affinity PipX partners.
In S. elongatus pipX is co-transcribed with the downstream gene pipY. This last gene belongs to the widely distributed and highly conserved pyridoxal phosphate (PLP)-binding protein (COG0325/PLPBP) family that is involved in vitamin B6 and amino acid homeostasis [19]. The PLPBP family (also termed ProsC/PROSC or COG0325 family) members are found in all kingdoms of life, exemplified by the proteins YBL036C (yeast), YggS (Gram-negative bacteria), YlmE (Gram-positive bacteria), PipY (cyanobacteria), PLPHP (humans) and HTH5 (rice). These are all single-domain proteins exhibiting the fold type III of PLP-holoenzymes [20,21,22,23,24] with no known enzymatic activity.

2. Structural and Functional Features of PLPBPs

2.1. PLP Is Solvent-Exposed in PLPBP Structures

The vitamin B6 vitamer PLP is used as a cofactor for enzyme-catalyzed reactions which include transamination, decarboxylation, racemization, aldol cleavage, or replacement reactions among others [26]. Since amino acid metabolism and other essential processes require PLP-dependent enzymes [27,28], PLP availability is of paramount importance to supply cofactors to activate newly synthesized apo-B6 enzymes. PLP is also required as a cofactor of glycogen phosphorylase [29] and certain transcriptional factors and regulators [28]. However, its aldehyde group endows PLP with high chemical reactivity, sometimes causing the inactivation of proteins (see for example, [30]), and therefore additional mechanisms are required for keeping the levels of free PLP low in cells and tissues. In the first report of a member of this family, Eswaramoorthy et al. (2003) documented structural parallelisms between the yeast protein YBL036C and the N-terminal domain of alanine racemases, leading them to infer (and even to provide some experimental hints for it) that PLPBP had alanine racemase activity [21]. However, no amino acid racemase, decarboxylase, deaminase, or transaminase activities were found for E. coli or human proteins [31], and although crystal structures of alanine racemase with bound substrates (D-ala) or inhibitors (D-cycloserine) have been determined [32], extensive crystallization attempts with these molecules did not detect any binding to PipY [22]. Furthermore, in vivo work did not support alanine racemase activity for S. elongatus PipY [19]. Therefore, despite the key importance of the PLP cofactor for PLPBP function (see below), PLP appears to have no catalytic function in the PLPBP family. Structures of six PLPBP members have been determined and deposited in the Protein DataBank (PDB, “https://www.rcsb.org/ (accesed on 1 September 2022)”) (Table 1). All of these structures correspond to single-domain chains folded according to the triose phosphate isomerase (TIM) barrel typically found in the fold type III of PLP-dependent enzymes. The only ones reported to date from a eukaryotic organism correspond to yeast protein YBL036C. The others are from a Gram-positive bacterium (Bifidobacterium adolescentis), and four Gram-negative bacteria including the cyanobacterium S. elongatus (Table 1). S. elongatus PipY structures with and without PLP offer high resolution and have been used to estimate the effects of clinical missense mutations found in the PLPBP human gene in patients with vitamin B6-dependent epilepsy [22,23]. Here, we use PipY as a reference for the additional discussion on structural and functional details concerning studied members of the protein family. Figure 1 shows the structure of PipY containing PLP (PDB file 5NM8).
Figure 1. Structure of PipY from S. elongatus (PDB 5NM8) colored for the evolutionary conservation of residues among PLPBP homologs, and mapping therein, residues targeted by missense mutations. The structure is in a cartoon representation except for the PLP, which is in a stick representation with C, O, N, and P atoms in yellow, red, blue, and orange, respectively. Color-coding of the structure from cyan to magenta according to the residue conservation score (the higher, the more conserved) given by The ConSurf Server “URL https://consurf.tau.ac.il/consurf_index.php (accesed on 3 August 2022) when queried with chain A of the PDB 5NM8, with default parameters. Spheres mark the location in PipY of known human PLPHP mutations (see Table 2). Residue numbers are given in one letter code, in black for S. elongatus, and shown in red, green, and blue, the human mutations causing vitamin B6-dependent epilepsy, and the in vitro mutations obtained in the corresponding proteins of F. nucleatum, and E. coli, respectively.
Table 1. Structures of COG0325/PLPB family proteins were determined and deposited in the Protein DataBank (PDB).