Characterization of the Escherichia coli pyridoxal 5′‐phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability

Abstract

The pyridoxal 5′‐phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP‐dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP‐dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.

Two PYRIDOXAL PHOSPHATE HOMEOSTASIS PROTEINs are essential for management of the coenzyme in plants

“…Thus, both of the Arabidopsis PLPHPs have the capacity to bind PLP. They behave as monomers with PLP likely solvent exposed similar to the reported oligomeric state of bacterial and yeast homologs (Eswaramoorthy et al, 2003;Prunetti et al, 2016;Tremiño et al, 2017;Tramonti et al, 2022), although the human ortholog is reported to exist as both monomers and dimers in vitro (Tremiño et al, 2018;Fux and Sieber, 2020). Significantly, the monomeric state of Arabidopsis PLPHPs contrasts with reported PLP-dependent enzymes from all kingdoms, all of which have higher oligomeric states (Eliot and Kirsch, 2004).…”

“…Expression and purification of the AtPLPHP1 K55A mutant followed by spectral analysis indicated an at least 10-fold lower amount of bound PLP, thus supporting the involvement of K55 in Schiff’s base formation with the coenzyme (Figure 2B). Although, it should be noted that a second lysine not involved in Schiff’s base formation with PLP but required for functionality as demonstrated with the E. coli homolog (Tramonti et al, 2022) is conserved in the Arabidopsis orthologs (Figure 2A).…”

“…As PLPHP has not been studied in plants, we performed a systematics analysis to establish homologs in plantae . At the time of the analysis, 8345 genes from 7793 taxa were predicted to be orthologous to the Escherichia coli PLPHP homolog (named YggS) (Ito et al, 2013; Prunetti et al, 2016; Tramonti et al, 2022) under the K06997 identifier at the KEGG orthology database (https://www.genome.jp/kegg/ko.html)(Kanehisa et al, 2016). The list included 317 orthologs from 142 plant taxa.…”

“…Similar to PLPHPs characterized from other kingdoms, the plant homologs would be predicted to bind PLP. Indeed, out of the 22 amino acid residues forming the PLP binding pocket (L32, A33, V34, S35, K36, K38, G55, E56, N57, Y58, I82, G83, P84, M164, I166, G203, M204, S205, R220, I221, G222, and T223) in the well-characterized homolog YggS from E. coli (Tramonti et al, 2022), 15 showed complete conservation in both homologs of core Angiosperms (Figure 2A). In Arabidopsis, 16 residues are conserved in both homologs, annotated here as AtPLPHP1 (At1g11930) and AtPLPHP2 (At4g26860), which in turn share 71% identity (Figure 2A).…”

Pleiotropic effects of PipX, PipY, or RelQ overexpression on growth, cell size, photosynthesis, and polyphosphate accumulation in the cyanobacterium Synechococcus elongatus PCC7942

“…In particular, the phenotypic features described here for PipY ox cultures should help studies on the structure–function relationship at PipY/PLPBP or concerning the regulatory complexities of accumulation of giant polyP granules in S. elongatus . For instances, PipY ox derivatives carrying mutations at functionally relevant or conserved residues ( Tramonti et al, 2022 ; Tremiño et al, 2022 ) can be easily compared in the presence of IPTG and further analyzed for traits of interest.…”