Mitochondrial phosphatase PTPMT1 is essential for cardiolipin biosynthesis

PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that?whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1 deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in?vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.     

Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts

Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli; phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of E. coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M-H](-) ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites.     

Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens

A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.     

Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis

The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe²⁺/α-KG/O₂ dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo₂-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo₂-lipid IV_A or Kdo₂-lipid A as a substrate, but not Kdo-lipid IV_Ain vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis.     

Enzymatic sorting of bacterial colonies on filter paper replicas: detection of labile activities.

To utilize autoradiographic colony-sorting techniques (C. R. H. Raetz, Proc. Natl. Acad. Sci. U.S.A. 72:2274-2278, 1975) for the isolation of mutants with unstable enzymes, we report a new desiccation-induced lysis method, compatible with low temperatures. Furthermore, a general, two-step protocol is presented for clonal detection of hydrolytic reactions. The advantages of these critical modifications are demonstrated with the membrane enzymes glycerol 3-phosphate acyltransferase and cytidine 5'-diphosphate-diglyceride hydrolase.     

Assignment of 1H, 13C and 15N backbone resonances of Escherichia coli LpxC bound to L-161,240

The UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A biosynthesis, an essential pathway in Gram-negative bacteria. We report the backbone resonance assignments of the 34 kDa LpxC from Escherichia coli in complex with the antibiotic L-161,240 using multidimensional, multinuclear NMR experiments. The ¹H chemical shifts of complexed L-161,240 are also determined.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

The serendipitous origin of chordate secretin peptide family members

Background  

The secretin family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (secretin family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic.     

An inner membrane dioxygenase that generates the 2-hydroxymyristate moiety of Salmonella lipid A

The lipid A residues of certain Gram-negative bacteria, including most strains of Salmonella and Pseudomonas, are esterified with one or two secondary S-2-hydroxyacyl chains. The S-2 hydroxylation process is O 2-dependent in vivo, but the relevant enzymatic pathways have not been fully characterized because in vitro assays have not been developed. We previously reported that expression of the Salmonella lpxO gene confers upon Escherichia coli K-12 the ability to synthesize 2-hydroxymyristate modified lipid A ( J. Biol. Chem. (2000) 275, 32940-32949). We now demonstrate that inactivation of lpxO, which encodes a putative Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase, abolishes S-2-hydroxymyristate formation in S. typhimurium. Membranes of E. coli strains expressing lpxO are able to hydroxylate Kdo 2-[4'- (32)P]-lipid A in vitro in the presence of Fe (2+), O 2, alpha-ketoglutarate, ascorbate, and Triton X-100. The Fe (2+) chelator 2,2'-bipyridyl inhibits the reaction. The product generated in vitro is a monohydroxylated Kdo 2-lipid A derivative. The [4'- (32)P]-lipid A released by mild acid hydrolysis from the in vitro product migrates with authentic S-2-hydroxlyated lipid A isolated from (32)P-labeled S. typhimurium cells. Electrospray ionization mass spectrometry and gas chromatography/mass spectrometry of the in vitro product are consistent with the 2-hydroxylation of the 3'-secondary myristoyl chain of Kdo 2-lipid A. LpxO contains two predicted trans-membrane helices (one at each end of the protein), and its active site likely faces the cytoplasm. LpxO is an unusual example of an integral membrane protein that is a member of the Fe (2+)/O 2/alpha-ketoglutarate-dependent dioxygenase family.     

Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis

LpxD catalyzes the third step of lipid A biosynthesis, the (R)-3-hydroxymyristoyl-acyl carrier protein ( R-3-OHC14-ACP)-dependent N-acylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine [UDP-3-O-(R-3-OHC14)-GlcN]. We have now overexpressed and purified Escherichia coli LpxD to homogeneity. Steady-state kinetics suggest a compulsory ordered mechanism in which R-3-OHC14-ACP binds prior to UDP-3-O-(R-3-OHC14)-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC14-ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC14)-GlcN. UDP-2-N-[(R)-3-Hydroxymyristoyl]-alpha-D-glucosamine, obtained by mild base hydrolysis of UDP-2,3-diacylglucosamine, is a noncompetitive inhibitor against both substrates. Synthetic (R)-3-hydroxylauroyl-methylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC14-ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC14)-GlcN, but (R)-3-hydroxylauroyl-methylphosphopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC14-ACP has a high binding affinity for free LpxD whereas UDP-3-O-(R-3-OHC14)-GlcN does not. Divalent cations inhibit R-3-OHC14-ACP-dependent acylation but not (R)-3-hydroxylauroyl-methylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC14-ACP contributes to binding. The F41A mutation increases the K(M) for UDP-3-O-(R-3-OHC14)-GlcN 30-fold, consistent with aromatic stacking of the corresponding F43 side chain against the uracil moiety of bound UDP-GlcNAc in the X-ray structure of Chlamydia trachomatis LpxD. Mutagenesis implicates E. coli H239 but excludes H276 as the catalytic base, and neither residue is likely to stabilize the oxyanion intermediate.