Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis

Abstract

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.

Undecaprenyl phosphate metabolism in Gram-negative and Gram-positive bacteria

“…The construction of fusion proteins of truncated YbjG and alkaline phosphatase (PhoA), and also truncated LpxT and PhoA, confirms this orientation [38]. The construction of truncated forms of PgpB and BlaM hybrid protein demonstrates that the substrate binding site of PgpB is also oriented to the periplasmic side of the inner membrane [35], and the recently solved crystal structures of PgpB from E. coli (accession code in the Protein Data Bank; 5JWY) [39] and YodM from B. subtilis (accession code in the Protein Data Bank; 5JKI) [37] also indicates the substrate binding site is oriented outside. Thus, all PAP2 enzymes catalyze the reaction on the periplasmic side of the membrane.…”

“…LpxT seems to be a phosphotransferase rather than a phosphatase in vivo [31,36]. Purified B. subtilis YodM efficiently catalyzes the dephosphorylation of phosphatidylglycerol phosphate [37]. It shows lower activity to dephosphorylate UPP than that to dephosphorylate FPP and IPP, and shows no activity to dephosphorylate phosphatidic acid.…”

“…Two distinct groups of membrane-integrated phosphatases, BacA homologues [26][27][28][29][30] and type-2 phosphatidic acid phosphatase (PAP2) homologues [31][32][33][34][35][36][37][38][39], catalyze the dephosphorylation of UPP in bacterial cells. The substrate specificity and the substrate binding site in the membrane have been the primary topics of interest to elucidate the physiological role of these enzymes because the substrate, UPP, is produced at both sides of the membrane in the process of polysaccharide synthesis.…”

In situ serial crystallography for rapid de novo membrane protein structure determination

“…Four proteins were used in this study as follows: the peptide transporter, PepT St , from Streptococcus thermophilus 37 , the lipoprotein signal peptidase II, LspA, from Pseudomonas aeruginosa (PAO1) 38 , the undecaprenyl-pyrophosphate phosphatase, BacA, from Escherichia coli (K-12) 39 , and the phosphatidic acid phosphatase, PgpB, from Bacillus subtilis 40 . Se-LspA, PgpB, BacA, and native S-PepT St were produced recombinantly and purified from biomass following published protocols 37–40 .…”

“…Se-LspA, PgpB, BacA, and native S-PepT St were produced recombinantly and purified from biomass following published protocols 37–40 . Se-PepT St was produced by using E. coli C43 (DE3) (NEB) cells with the pWaldo- dtpT plasmid.…”