Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitine by Escherichia coli

Two proteins (CaiB and CaiD) were found to catalyze the reversible biotransformation of crotonobetaine to L-carnitine in Escherichia coli in the presence of a cosubstrate (e.g., gamma-butyrobetainyl-CoA or crotonobetainyl-CoA). CaiB (45 kDa) and CaiD (27 kDa) were purified in two steps to electrophoretic homogeneity from overexpression strains. CaiB was identified as crotonobetainyl-CoA:carnitine CoA-transferase by MALDI-TOF mass spectrometry and enzymatic assays. The enzyme exhibits high cosubstrate specificity to CoA derivatives of trimethylammonium compounds. In particular, the N-terminus of CaiB shows significant identity with other CoA-transferases (e.g., FldA from Clostridium sporogenes, Frc from Oxalobacter formigenes, and BbsE from Thauera aromatica) and CoA-hydrolases (e.g., BaiF from Eubacterium sp.). CaiD was shown to be a crotonobetainyl-CoA hydratase using MALDI-TOF mass spectrometry and enzymatic assays. Besides crotonobetainyl-CoA CaiD is also able to hydrate crotonyl-CoA with a significantly lower Vmax (factor of 10(3)) but not crotonobetaine. The substrate specificity of CaiD and its homology to the crotonase confirm this enzyme as a new member of the crotonase superfamily. Concluding these results, it was verified that hydration of crotonobetaine to L-carnitine proceeds at the CoA level in two steps: the CaiD catalyzed hydration of crotonobetainyl-CoA to L-carnitinyl-CoA, followed by a CoA transfer from L-carnitinyl-CoA to crotonobetaine, catalyzed by CaiB. When gamma-butyrobetainyl-CoA was used as a cosubstrate (CoA donor), the first reaction is the CoA transfer. The optimal ratios of CaiB and CaiD during this hydration reaction, determined to be 4:1 when crotonobetainyl-CoA was used as cosubstrate and 5:1 when gamma-butyrobetainyl-CoA was used as cosubstrate, are different from that found for in vivo conditions (1:3).     

Biotransformation of crotonobetaine to L(–)-carnitine in Proteus sp.

Two proteins, component I (CI) and component II (CII), catalyze the biotransformation of crotonobetaine to L(-)-carnitine in Proteus sp. CI was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. The N-terminal amino acid sequence of CI showed high similarity (80%) to the caiB gene product from Escherichia coli O44K74, which encodes the L(-)-carnitine dehydratase. CI alone was unable to convert crotonobetaine into L(-)-carnitine even in the presence of the cosubstrates crotonobetainyl-CoA or gamma-butyrobetainyl-CoA, which are essential for this biotransformation. The relative molecular mass of CI was determined to be 91.1 kDa. CI is composed of two identical subunits of molecular mass 43.6 kDa. The isoelectric point is 5.0. CII was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. and its N-terminal amino acid sequence showed high similarity (75%) to the caiD gene product of E. coli O44K74. The relative molecular mass of CII was shown to be 88.0 kDa, and CII is composed of three identical subunits of molecular mass 30.1 kDa. The isoelectric point of CII is 4.9. For the biotransformation of crotonobetaine to L(-)-carnitine, the presence of CI, CII, and a cosubstrate (crotonobetainyl-CoA or gamma-butyrobetainyl-CoA) were shown to be essential.     

Identification and functional characterisation of genes and corresponding enzymes involved in carnitine metabolism of <Emphasis Type="Italic">Proteus</Emphasis> sp.

Enzymes involved in carnitine metabolism of Proteus sp. are encoded by the cai genes organised as the caiTABCDEF operon. The complete operon could be sequenced from the genomic DNA of Proteus sp. Amino acid sequence similarities and/or enzymatic analysis confirmed the function assigned to each protein involved in carnitine metabolism. CaiT was suggested to be an integral membrane protein responsible for the transport of betaines. The caiA gene product was shown to be a crotonobetainyl-CoA reductase catalysing the irreversible reduction of crotonobetainyl-CoA to -butyrobetainyl-CoA. CaiB and CaiD were identified to be the two components of the crotonobetaine hydrating system, already described. CaiB and caiD were cloned and expressed in Escherichia coli. After purification of both proteins, their individual enzymatic functions were solved. CaiB acts as betainyl-CoA transferase specific for carnitine, crotonobetaine, -butyrobetaine and its CoA derivatives. Transferase reaction proceeds, following a sequential bisubstrate mechanism. CaiD was identified to be a crotonobetainyl-CoA hydratase belonging to the crotononase superfamily. Because of amino acid sequence similarities, CaiC was suggested to be a betainyl-CoA ligase. Taken together, these results show that the metabolism of carnitine and crotonobetaine in Proteus sp. proceeds at the CoA level.     

Crotonobetaine reductase from Escherichia coli consists of two proteins

Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)). CI has been purified to electrophoretic homogeneity from a cell-free extract of E. coli O44 K74. The purified protein shows l(-)-carnitine dehydratase activity and its N-terminal amino acid sequence is identical to the caiB gene product from E. coli O44 K74. The relative molecular mass of CI has been determined to be 86100. It is composed of two identical subunits with a molecular mass of 42600. The isoelectric point of CI was found to be 4.3. CII was purified from an overexpression strain in one step by ion exchange chromatography on Fractogel EMD TMAE 650(S). The N-terminal amino acid sequence of CII shows absolute identity with the N-terminal sequence of the caiA gene product, i.e. of the postulated crotonobetaine reductase. The relative molecular mass of the protein is 164400 and it is composed of four identical subunits of molecular mass 41500. The isoelectric point of CII is 5.6. CII contains non-covalently bound FAD in a molar ratio of 1:1. In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII. A still unknown low-molecular-mass effector described for the l(-)-carnitine dehydratase is also necessary for crotonobetaine reductase activity. Monoclonal antibodies were raised against the two components of crotonobetaine reductase.     

The tetrahydropyranopterin structure of the sulfur-free and metal-free molybdenum cofactor precursor

The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase. Moco is synthesized by a unique and evolutionary old multi-step pathway with two intermediates identified so far, the sulfur-free and metal-free pterin derivative precursor Z and molybdopterin, a pterin with an enedithiolate function essential for Mo ligation. The latter pterin component is believed to form a tetrahydropyranopterin similar to the one found for Moco in the crystal structure of Mo as well as tungsten (W) enzymes. Here we report the spectroscopic characterization and structure elucidation of precursor Z purified from Escherichia coli overproducing MoaA and MoaC, two proteins essential for bacterial precursor Z synthesis. We have shown that purified precursor Z is as active as precursor Z present in E. coli cell extracts, demonstrating that no modifications during the purification procedure have occurred. High resolution electrospray ionization mass spectrometry afforded a [M + H]+ ion compatible with a molecular formula of C10H15N5O8P. Consequently 1H NMR spectroscopy not allowed structural characterization of the molecule but confirmed that this intermediate undergoes direct oxidation to the previously well characterized non-productive follow-up product compound Z. The 1H chemical shift and coupling constant data are incompatible with previous structural proposals and indicate that precursor Z already is a tetrahydropyranopterin system and carries a geminal diol function in the C1' position.     

Purification and characterisation of the BIOH protein from the biotin biosynthetic pathway

Conversion of pimeloyl-coenzyme A (CoA) to biotin in Escherichia coli requires at least four enzymes encoded by genes in the bio operon. One gene, bioH, which is not present in the bioABFCD operon, is required for the synthesis of pimeloyl-CoA but its exact role in formation of this intermediate is unknown. To investigate this further, we have overexpressed and purified the bioH gene products from both E. coli (BIOH EC) and Neisseria meningitis (BIOH NM) in E. coli. When purified BIOH was incubated with excess CoA and analysed by electrospray mass spectrometry a species of mass corresponding to a BIOH:CoA complex was observed. Mutation of a conserved serine residue to alanine (BIOH EC S82A) did not prevent CoA binding. This is the first report of the purification of BIOH and the observation of a small molecule bound to the protein provides clues to its role in pimeloyl-CoA synthesis.     

Role of betaine:CoA ligase (CaiC) in the activation of betaines and the transfer of coenzyme A in Escherichia coli

Aims:  Characterization of the role of CaiC in the biotransformation of trimethylammonium compounds into l (−)-carnitine in Escherichia coli .
Methods and Results:  The caiC gene was cloned and overexpressed in E. coli and its effect on the production of l (−)-carnitine was analysed. Betaine:CoA ligase and CoA transferase activities were analysed in cell free extracts and products were studied by electrospray mass spectrometry (ESI-MS). Substrate specificity of the caiC gene product was high, reflecting the high specialization of the carnitine pathway. Although CoA-transferase activity was also detected in vitro , the main in vivo role of CaiC was found to be the synthesis of betainyl-CoAs. Overexpression of CaiC allowed the biotransformation of crotonobetaine to l (−)-carnitine to be enhanced nearly 20-fold, the yield reaching up to 30% (with growing cells). Higher yields were obtained using resting cells (up to 60%), even when d (+)-carnitine was used as substrate.
Conclusions:  The expression of CaiC is a control step in the biotransformation of trimethylammonium compounds in E. coli .
Significance and Impact of the Study:  A bacterial betaine:CoA ligase has been characterized for the first time, underlining its important role for the production of l -carnitine with Escherichia coli .     

Crystal structure of Escherichia coli crotonobetainyl-CoA: carnitine CoA-transferase (CaiB) and its complexes with CoA and carnitinyl-CoA

L-Carnitine (R-[-]-3-hydroxy-4-trimethylaminobutyrate) is found in both eukaryotic and prokaryotic cells and participates in diverse processes including long-chain fatty-acid transport and osmoprotection. The enzyme crotonobetainyl/gamma-butyrobetainyl-CoA:carnitine CoA-transferase (CaiB; E.C. 2.8.3.-) catalyzes the first step in carnitine metabolism, leading to the final product gamma-butyrobetaine. The crystal structures of Escherichia coli apo-CaiB, as well as its Asp169Ala mutant bound to CoA and to carnitinyl-CoA, have been determined and refined to 1.6, 2.4, and 2.4 A resolution, respectively. CaiB is composed of two identical circular chains that together form an intertwined dimer. Each monomer consists of a large domain, containing a Rossmann fold, and a small domain. The monomer and dimer resemble those of formyl-CoA transferase from Oxalobacter formigenes, as well as E. coli YfdW, a putative type-III CoA transferase of unknown function. The CoA cofactor-binding site is formed at the interface of the large domain of one monomer and the small domain from the second monomer. Most of the protein-CoA interactions are formed with the Rossmann fold domain. While the location of cofactor binding is similar in the three proteins, the specific CoA-protein interactions vary somewhat between CaiB, formyl-CoA transferase, and YfdW. CoA binding results in a change in the relative positions of the large and small domains compared with apo-CaiB. The observed carnitinyl-CoA product in crystals of the CaiB Asp169Ala mutant cocrystallized with crotonoyl-CoA and carnitine could result from (i) a catalytic mechanism involving a ternary enzyme-substrate complex, independent of a covalent anhydride intermediate with Asp169, (ii) a spontaneous reaction of the substrates in solution, followed by binding to the enzyme, or (iii) an involvement of another residue substituting functionally for Asp169, such as Glu23.     

Salt stress effects on the central and carnitine metabolisms of Escherichia coli

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.     

Isolation, primary structure and synthesis of heat-stable enterotoxin produced by Yersinia enterocolitica

Five heat-stable enterotoxins were isolated from the culture supernatant of Yersinia enterocolitica and purified to homogeneity by DEAE-Sephacel and high-performance liquid chromatographies. They caused acute fluid accumulation in the intestine of suckling mice. The amino acid sequence of one of the enterotoxins was determined to be Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys, by Edman degradation of its pyridylethylated derivative and a combination of fast atom bombardment mass spectrometry and carboxypeptidase B digestion. This structure was unambiguously confirmed by chemical synthesis. The other enterotoxins had longer or shorter amino acid sequences at their N termini, but the same sequence at their C termini. The six half-cystine residues formed intramolecular disulfide linkages, as shown by measurement of the molecular masses of the enterotoxins by fast atom bombardment mass spectrometry. The sequence of 13 amino acid residues at the C terminus showed similarity to those of heat-stable enterotoxins isolated from enterotoxigenic Escherichia coli [Aimoto, S. et al. (1982) Eur. J. Biochem. 129, 257-263; Takao, T. et al. (1983) FEBS Lett. 152, 1-5] suggesting that these similar sequences are related to the common biological and immunological properties of enterotoxins produced by Y. enterocolitica and enterotoxigenic E. coli.     

Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia coli. Purification by high performance liquid chromatography and direct analysis by plasma desorption mass spectrometry

Lipopolysaccharide (LPS) extracted from the deep rough mutant of Escherichia coli D31m4 was disaggregated with 0.1 M EDTA, pH 7.0, and fractionated on a diethylaminoethyl-cellulose column to yield the biphosphate form of LPS. After methylation, the derivative was purified by reverse-phase high performance liquid chromatography using a C18-bonded silica cartridge. A linear gradient of 50-100% isopropyl alcohol/water (93:7, v/v) in acetonitrile/water (93:7, v/v) was used over a period of 60 min. The derivatized LPS showed a single major peak by high performance liquid chromatography, and this hexamethyl hexaacyl LPS was recovered and subjected to chemical analysis, plasma desorption mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. Chemical analysis of the purified hexamethyl LPS quantitated certain key chemical compositions. Plasma desorption mass spectrometry showed a molecular ion (M + CH2 + Na)+ at m/z 2360, which established the molecular formula and Mr to be C116H214N2O39P2 and 2323, respectively. Thus, it contained two each of glucosamine, 2-keto-3-deoxyoctonate, and phosphate; four beta-hydroxymyristates; one laurate; and one myristate. NMR spectroscopy confirmed the locations of the four ester-linked fatty acyl groups. Based on these results and the known structure of free lipid A, the complete structure of the deep-rough chemotype LPS from E. coli can now be presented with confidence. This is the first report of a successful purification to homogeneity and the characterization of the simplest of the LPS at the intact level. This study shows that the natural distribution of the lipid A moiety of LPS from E. coli D31m4 is hexaacyl/pentaacyl in a molar ratio of greater than 90:less than 10. Acid hydrolysis of LPS causes the formation of the lower homologues of the free lipid A.     

Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia)

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.     

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.     

Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites

Pantothenate is the precursor of the essential cofactor coenzyme A (CoA). Pantothenate kinase (CoaA) catalyzes the first and regulatory step in the CoA biosynthetic pathway. The pantothenate analogs N-pentylpantothenamide and N-heptylpantothenamide possess antibiotic activity against Escherichia coli. Both compounds are substrates for E. coli CoaA and competitively inhibit the phosphorylation of pantothenate. The phosphorylated pantothenamides are further converted to CoA analogs, which were previously predicted to act as inhibitors of CoA-dependent enzymes. Here we show that the mechanism for the toxicity of the pantothenamides is due to the inhibition of fatty acid biosynthesis through the formation and accumulation of the inactive acyl carrier protein (ACP), which was easily observed as a faster migrating protein using conformationally sensitive gel electrophoresis. E. coli treated with the pantothenamides lost the ability to incorporate [1-(14)C]acetate to its membrane lipids, indicative of the inhibition of fatty acid synthesis. Cellular CoA was maintained at the level sufficient for bacterial protein synthesis. Electrospray ionization time-of-flight mass spectrometry confirmed that the inactive ACP was the product of the transfer of the inactive phosphopantothenamide moiety of the CoA analog to apo-ACP, forming the ACP analog that lacks the sulfhydryl group for the attachment of acyl chains for fatty acid synthesis. Inactive ACP accumulated in pantothenamide-treated cells because of the active hydrolysis of regular ACP and the slow turnover of the inactive prosthetic group. Thus, the pantothenamides are pro-antibiotics that inhibit fatty acid synthesis and bacterial growth because of the covalent modification of ACP.     

Acyl carrier protein synthases from gram-negative, gram-positive, and atypical bacterial species: Biochemical and structural properties and physiological implications

Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae. Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology.     

Neue ent-atisiren- und ent-kaurensäure-derivate aus Helianthus-arten

The investigation of four Helianthus species afforded, in addition to known compounds, four new atisirenic acid derivatives, a new ent-kaurenic acid derivative, three new labdane derivatives and an unknown C₁₅-acetylenic compound. The structures of the diterpenes were elucidated by intensive NMR studies and some chemical transformations. An unusual fragmentation in the mass spectra of the atisirenes is discussed briefly.     

Reexamination of the 1H NMR assignments and solution conformations of L-carnitine and O-acetyl-L-carnitine using C-2 stereospecifically labeled monodeuterated isomers

The two C-2 monodeuterated isomers of L-carnitine were synthesized by enzymatic hydration of crotonobetaine in D2O and by enzymatic proton exchange of L-[2-2H2]carnitine in H2O. These reactions, catalyzed by an induced Escherichia coli carnitine hydrolyase proceed stereospecifically. The two isomers of L-[2-2H]carnitine were examined by 1H NMR at 500 MHz, which allowed us to independently monitor the pD dependence and coupling constants of the H-2 protons. The results obtained indicate that there is little effect of the carboxyl charge on the conformational state(s) of L-carnitine about the C-2/C-3 bond. The NMR data obtained in this study do not support previous solution studies of the pH-dependent conformational changes for DL-carnitine nor the proposed conformation of O-acetyl-DL-carnitine in the crystalline state.     

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.

Two-thirds of the lipid A in wild-type Escherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'. The remaining lipid A contains a monophosphate substituent at position 4' and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 degrees C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-L-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.     

Purification and properties of carnitine dehydratase from Escherichia coli--a new enzyme of carnitine metabolization

Carnitine dehydratase from Escherichia coli 044 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine. It has been purified 500-fold to electrophoretic homogeneity by chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, second phenyl-Sepharose and finally gel filtration on a Sephadex G-100 column. During the purification procedure a low-molecular-weight effector essential for enzyme activity was separated from the enzyme. The addition of this still unknown effector caused reactivation of the apoenzyme. The relative molecular mass of the apoenzyme has been estimated to be 85,000. It seems to be composed of two identical subunits with a relative molecular mass of 45,000. The purified and reactivated enzyme has been further characterized with respect to pH and temperature optimum (7.8 and 37-42 degrees C), equilibrium constant (Keq = 1.5 +/- 0.2) and substrate specifity. The enzyme is inhibited by thiol reagents. The Km value for crotonobetaine is 1.2.10(-2) M. gamma-Butyrobetaine, D(+)-carnitine and choline are competitive inhibitors of crotonobetaine hydration.