9.B.105 The Peptidase/Phosphatase/Lead Resistance Fusion Protein (PPLR) Family Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Ralstonia (Cupriavidus) metallidurans CH34 to Pb²⁺(Taghavi et al., 2009). Structural Pb²⁺-resistance genes of the pMOL30-encoded pbrUTRABCD operon form the major line of defense to Pb²⁺(Taghavi et al., 2009). The expression of the pbrR(2) cadA pbrC(2) operon of the CMGI-1 region and the chromosomal zntA gene were clearly induced in the presence of Pb²⁺. After inactivation of the pbrA, pbrB or pbrD gene, expression of the pbrR(2) cadA pbrC(2) operon went up considerably. This points towards synergistic interactions between pbrUTRABCD and pbrR(2) cadA pbrC(2) to maintain a low intracellular Pb²⁺concentration, where pbrR(2) cadA pbrC(2) gene functions can complement and compensate for mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement the loss of pbrA was further confirmed by mutation analysis (Taghavi et al., 2009). The pbrB::Tn(Km2) mutation resulted in the most significant decrease of Pb²⁺resistance, indicating that Pb²⁺sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in pbr-encoded Pb²⁺resistance. Residues 1-190 ( TMSs 1-6) in PbrB/C code for a PAP2-type protein in a family of phosphatases and haloperoxidases. These homologues are found only in bacteria and may be integral membrane phospholipid phosphatases. The C-terminal residues (residues 200-340; TMSs 7-10) may code for a signal peptidase II (pfam01252). In fact, several member of this family are annotated as lipoprotein signal peptidases, suggesting that these integral membrane proteins may be proteases. Other members (see subfamilies 4 - 6; subfamilies 5 and 6 were formerly subfamilies 9.B.196.1 and 2, respectively) appear to belong to the Phosphodiester PA Phosphatase (PAP2) Superfamily (Sigal et al. 2005). The PAP2 superfamily consists of numerous integral membrane proteins, annotated in Pfam and NCBI as phosphodiesterases. Phosphatidylcholine (PC) hydrolysis generates two second messengers: phosphatidic acid (PA) and diacylglycerol (DAG). Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAPase) are involved in their generation and therefore are key enzymes in signal transduction. Specific isoforms of these enzymes are activated by receptor occupancy in the brain. Phosphatidylinositol 4,5-bisphosphate-dependent PLD (PIP2-PLD) and N-ethylmaleimide-insensitive PAPase (PAP2) have been suggested to act in series to generate the biologically active lipids PA and DAG (Salvador et al. 2002). PbrBC may be a phosphatase. While PbrA non-specifically exported Pb²⁺, Zn²⁺ and Cd²⁺, a specific increase in lead resistance is observed when PbrA and PbrB are coexpressed. Possibly Pb²⁺ is exported from the cytoplasm by PbrA and then sequestered as a phosphate salt with the inorganic phosphate produced by PbrB. Similar operons containing genes for heavy metal translocating ATPases and phosphatases can be found in many different bacterial species, suggesting that lead detoxification through active efflux and sequestration is a common lead-resistance mechanism (Hynninen et al. 2009).
This family belongs to the Phospholipid Transporter/Phosphatase (PL-TP) Superfamily.
References:
Ghachi, M.E., N. Howe, R. Auger, A. Lambion, A. Guiseppi, F. Delbrassine, G. Manat, S. Roure, S. Peslier, E. Sauvage, L. Vogeley, J.C. Rengifo-Gonzalez, P. Charlier, D. Mengin-Lecreulx, M. Foglino, T. Touzé, M. Caffrey, and F. Kerff. (2017). Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis. Cell Mol Life Sci. [Epub: Ahead of Print]
Hug, L.A., B.J. Baker, K. Anantharaman, C.T. Brown, A.J. Probst, C.J. Castelle, C.N. Butterfield, A.W. Hernsdorf, Y. Amano, K. Ise, Y. Suzuki, N. Dudek, D.A. Relman, K.M. Finstad, R. Amundson, B.C. Thomas, and J.F. Banfield. (2016). A new view of the tree of life. Nat Microbiol 1: 16048.
Hynninen, A., T. Touzé, L. Pitkänen, D. Mengin-Lecreulx, and M. Virta. (2009). An efflux transporter PbrA and a phosphatase PbrB cooperate in a lead-resistance mechanism in bacteria. Mol. Microbiol. 74: 384-394.
Moll, R.G. and G. Schäfer. (2004). Novel functional aspects of the membrane-bound exo-pyrophosphatase of the hyperthermoacidophilic archaeon Sulfolobus are provided by analysis of its gene and the adjacent gene cluster. J. Bioenerg. Biomembr. 36: 143-150.
Salvador, G.A., S.J. Pasquaré, M.G. Ilincheta de Boschero, and N.M. Giusto. (2002). Differential modulation of phospholipase D and phosphatidate phosphohydrolase during aging in rat cerebral cortex synaptosomes. Exp Gerontol 37: 543-552.
Sigal, Y.J., M.I. McDermott, and A.J. Morris. (2005). Integral membrane lipid phosphatases/phosphotransferases: common structure and diverse functions. Biochem. J. 387: 281-293.
Taghavi, S., C. Lesaulnier, S. Monchy, R. Wattiez, M. Mergeay, and D. van der Lelie. (2009). Lead(II) resistance in Cupriavidus metallidurans CH34: interplay between plasmid and chromosomally-located functions. Antonie Van Leeuwenhoek 96: 171-182.
Wu, W.I., Y. Liu, B. Riedel, J.B. Wissing, A.S. Fischl, and G.M. Carman. (1996). Purification and characterization of diacylglycerol pyrophosphate phosphatase from Saccharomyces cerevisiae. J. Biol. Chem. 271: 1868-1876.
Examples:
TC#	Name	Organismal Type	Example
9.B.105.1.1	PbrB/C fusion protein of 358 aas and 10 - 12 TMSs. Confers resistance to Pb²⁺ (Taghavi et al., 2009). May be a phosphatase. While PbrA non-specifically exported Pb²⁺, Zn²⁺ and Cd²⁺, a specific increase in lead resistance is observed when PbrA and PbrB are coexpressed. Possibly Pb²⁺ is exported from the cytoplasm by PbrA and then sequestered as a phosphate salt with the inorganic phosphate produced by PbrB. Similar operons containing genes for heavy metal translocating ATPases and phosphatases can be found in many different bacterial species, suggesting that lead detoxification through active efflux and sequestration is a common lead-resistance mechanism (Hynninen et al. 2009).	Bacteria	*PbrB/C fusion protein of Ralstonia (Cupriavidus) metallidurans* (Q58AJ7)**

9.B.105.1.10	Plasma-membrane-bound acidic pyrophosphatase (exo-PPase) of 206 aas and 5 TMSs. It represents proteins with the conserved phosphatase motif KxxxxxRP-(x12-54)-PSGH-(x31-54)-SRxxxxxHxxxD. Phosphatase or pyrophosphatase activity was described for the Sulfolobus acidocaldarius protein. It may function in resistance to the peptide antibiotic bacitracin (Moll and Schäfer 2004).		ExoPPase of Sulfolobus acidocaldarius

9.B.105.1.2	Hypothetical protein, HEAR1543. Residues 1-200 45% identity to PbrB which has 312 aas with 6 TMSs. The c-terminal domain contains a DUF3703 domain.	Bacteria	*HEAR1543 of Herminiimonas arsenicoxydans* (A4G5C1)**

9.B.105.1.3	Undecaprenyl-diphosphatase BcrC (EC 3.6.1.27) (Undecaprenyl pyrophosphate phosphatase)	Bacteria	bcrC of Bacillus subtilis

9.B.105.1.4	Hypothetical protein	Planctomycetes	HP of Rhodopirellula baltica

9.B.105.1.5	Lipoprotein signal peptidase of the Peptidase A8 Superfamily; IspA: geranyltranstransferase (Farnesyl-diphosphate synthase).	Firmicutes	Peptidase of Clostridium cellulolyticum

9.B.105.1.6	Putative phosphatase of 178 aas and 4 TMSs.	Spirochaetes	Putative phosphatase of Leptospira interrogans

9.B.105.1.7	Uncharacterized protein of 188 aas and 5 or 6 TMSs		UP of Bdellovibrio bacteriovorus

9.B.105.1.8	Lipoprotein signal peptidase of 310 aas and 6 TMSs, IspA.		IspA of Bdellovibrio bacteriovorus

9.B.105.1.9	*Uncharacterized protein of 141 aas and 4 TMSs (Hug et al.* 2016).**		UP of Candidatus Peribacter riflensis

Examples:
TC#	Name	Organismal Type	Example
9.B.105.2.1	Putative phosphatase of 321 aas and 9 TMSs	Spirochaetes	Putative phosphatase of Leptospira interrogans

9.B.105.2.2	Membrane-associated phospholipid phosphatase of 223 aas and 6 TMSs.		Phosphatase of Lactobacillus paracasei

9.B.105.2.3	*YodM (PgpB), the principal phosphatidylglycerol phosphate (PGP) phosphatase of 203 aas and 5 or 6 TMSs in B. subtilis. The crystal structure is known (Ghachi et al.* 2017).**		PgpB of Bacillus subtilis

9.B.105.2.4	Phosphatase PAP2 family proteinof 237 aas and 6 TMSs.		PAP2 family protein of Maritimibacter alkaliphilus

Examples:
TC#	Name	Organismal Type	Example
9.B.105.3.1	Putative phosphatidate phosphatase of 412 aas and 8 TMSs, Ppp		Ppp of Lucilia cuprina

9.B.105.3.2	Diacylglycerol pyrophosphate (DGPP) phosphatase catalyzes the dephosphorylation of diacylglycerol diphosphate (DGPP) to phosphatidate (PA) and the subsequent dephosphorylation of PA to diacylglycerol (DAG). It regulates intracellular DGPP and PA levels, which play a signaling role in stress responses. Can also use lysophosphatidic acid (LPA) and phosphatidylglycerophosphate as substrates. Substrate preference is DGPP > LPA > PA. Activity is independent of a divalent cation ion (Wu et al. 1996). The protein probably has 6 TMSs with two 3-TMS repeats.		*DGPP phosphatase of Saccharomyces cerevisiae* (Baker's yeast)**

9.B.105.3.3	Putative phosphatase of 223 aas and 5 TMSs.		Putative phosphatase of Bdellovibrio exovorus

9.B.105.3.4	Putative phospholipid phosphatase of 326 aas and 8 TMSs		PL phosphatase of Lactobacillus rhamnosis

9.B.105.3.5	*Highly similar to Homo sapiens* plasticity related gene 3 (PRG-3) of 301 aas and 5 or 6 putative TMSs.**		PRG3 of Homo sapiens

Examples:
TC#	Name	Organismal Type	Example
9.B.105.4.1	Uncharacterized protein of 217 aas and 8 TMSs in a 2 + 2 + 2 + 2 arrangement.		UP of Desulfitobacterium hafniense

9.B.105.4.2	Uncharacterized protein of 201 aas and 6 TMSs. Annotated as a phosphoesterase PA-phosphatase-like protein of the PAP2 superfamily.		PAP2 protein of Methanobacterium formicicum

9.B.105.4.3	Uncharacterized protein of 206 aas and 6 TMSs. Shows some sequence similarity with 9.A.29.3.2 (e-valute of 0.0008 over 118 aas).		UP of Streptomyces sulphureus

9.B.105.4.4	Putative uncharacterized phosphatase of 191 aas and 6 TMSs.		PAP2 family protein of Candidatus Beckwithbacteria bacterium

9.B.105.4.5	Putative phosphatase PAP2 family protein of 193 aas and 6 TMSs.		PAP2 family protein of Micromonospora wenchangensis

9.B.105.4.6	Uncharacterized protein of 230 aas and 6 putative TMSs		UP of Chitinophaga sp.

9.B.105.4.7	Phosphatase PAP2 family protein of 197 aas and 6 TMSs.		*PAP2 family protein of Rathayibacter* sp.**

Examples:
TC#	Name	Organismal Type	Example
9.B.105.5.1	PAP2 family phosphodiesterase of 239 aas and 5 TMSs.		PAP2 protein of Bdellovibrio exovorus

9.B.105.5.2	Putative phosphodiesterase of 323 aas and 4 - 6 TMSs.		Phosphodiesterase of Bacteroides ovatu

9.B.105.5.3	Phosphoesterase PA-phosphatase related protein of 234 aas and 6 probable TM		Phosphatase of Gemmatirosa kalamazoonesis

Examples:
TC#	Name	Organismal Type	Example
9.B.105.6.1	Uncharacterised protein of 308 aas and 8 TMSs		UP of Dermatophilus congolensis

9.B.105.6.2	Phosphatase PAP2 family protein of 357 aas and 8 TMSs.		PAP2 family protein of Bombiscardovia coagulans

9.B.105.6.3	Uncharacterized protein of 388 aas and 8 TMSs in a 1 + 5 + 2 arrangement.		UP of Tetrasphaera japonica

Examples:
TC#	Name	Organismal Type	Example