Pseudomonas aeruginosa is the causative agent of various opportunistic infections, including gut-derived sepsis. We previously demonstrated that the
serA gene is associated with the bacterial pathogenicity, and has a role in promoting the bacterial penetration through the Caco-2 cell monolayers, which was accompanied by decreased swimming and swarming motility, bacterial adherence, and fly mortality [
1]. Further, we previously investigated whether
l-serine, which is known to inhibit the
d-3-phosphoglycerate dehydrogenase (PGDH) activity of the SerA protein, significantly reduces the known phenotypes associated with bacterial pathogenicity. Consequently, the addition of
l-serine was found to significantly reduce the phenotypes associated with the bacterial pathogenicity, including bacterial penetration through Caco-2 cell monolayers, bacterial swimming and swarming motility, bacterial adherence, and fly mortality [
1]. Furthermore, we show that in a PGDH assay using crude extracts that were isolated from overnight cultures of
E.
coli overexpressing the
P.
aeruginosa serA gene,
l-serine directly inhibited the PGDH activity of the SerA protein. The background PGDH activity of the negative control strain was high, presumably due to contaminated proteins in the crude extracts. Therefore, to further confirm the direct inhibition of PGDH activity of
P.
aeruginosa SerA by
l-serine, we purified the PGDH from
P. aeruginosa using the glutathione S-transferase (GST) fusion protein system which is used for high-level expression and efficient purification of recombinant proteins.
As described in detail previously [
2,
3], the
serA gene encodes the
d-3-phosphoglycerate dehydrogenase (PGDH) and catalyzes the first step in serine synthesis by utilizing NAD
+ as a cofactor in
Escherichia coli. It is known that the activity of PGDHs derived from certain bacterial species including
E. coli, can be allosterically inhibited by
l-serine, the end product of the serine synthesis pathway, due to a conformational change in the three-dimensional structure of PGDH upon binding of
l-serine [
2,
3]. On the other hand, the activity of PGDHs isolated from rat and chicken livers appears not to be influenced by the addition of
l-serine [
4‐
6]. As described in detail previously [
7,
8], this difference in the inhibitory effects of
l-serine on the PGDH activity among these species seems to be dependent on the difference in the amino acid sequence and the three-dimensional structure of each PGDH. PGDHs consists of at least three different structural motifs that have been classified as types I, II, and III as described in detail previously [
7‐
10]. As described in detail previously [
6‐
8,
10], the PGDHs from certain bacteria including
E. coli, P. aeruginosa, H. influenza, and the simple eukaryotes such as yeast,
Leishmania, and
Neurospora, retain the type II motif, which contains three distinct domains called the cofactor or nucleotide-binding domain, the substrate-binding domain, and the C-terminal regulatory or the serine binding domain. The C-terminal regulatory or the serine binding domain is also called as the ACT (
aspartate kinase-
chorismate mutase-
tyrA prephenate dehydrogenase) domain and is responsible for
l-serine binding and the regulation of PGDH activity; in
E. coli PGDH, there are critical amino acid residues which are needed for
l-serine binding as described in detail previously [
7,
8]. Other bacteria, including
Mycobacterium tuberculosis,
Bacillus subtilis, and the higher eukaryotes, including mouse, rat, and human, possess the type I motif which harbors a large polypeptide insertion, which is called as ASB (
allosteric
substrate
binding) domain, in the C-terminal fragment which follows the substrate binding domain as described in detail previously [
7,
8]. Some organisms, including
Pyrococcus,
Rhodopseudomonas, Clostridium, Entamoeba histolytica, Bacteroides fragilis, and
Porphyromonas gingivalis, have the type III motif which lacks the C-terminal regulatory domain as described in detail previously [
7,
8]. Furthermore, there are two forms of the type III motif depending on whether lysine (type K) or histidine (type H) exists at the active site as described in detail previously [
7,
8,
10] .
In the present study, we performed the purification and characterization of the PGDH from P. aeruginosa PAO1 strain by exploiting the GST fusion protein system as there is no report yet available on the characterization of the P. aeruginosa PGDH. Furthermore, we determined the median inhibitory concentration (IC50) of d- and l-serine against the purified PGDH isolated from the P. aeruginosa serA gene.