Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.     

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.     

Phosphotransferase system of Staphylococcus aureus: its requirement for the accumulation and metabolism of galactosides

The phosphotransferase system of Staphylococcus aureus was characterized. Mutants defective in enzyme I and heat-stable (HPr) protein as well as in the two components specific to lactose accumulation, factor III and enzyme II, were isolated. Colorimetric assays for each of the components are presented based on the formation of o-nitrophenyl-beta-d-galactoside-6-phosphate by the system and its hydrolysis by the staphylococcal 6-phospho-beta-galactosidase. The components were partially purified and their molecular weights were estimated: enzyme I, 100,000 +/- 15%; HPr, 10,000 +/- 15%; factor III, 30,000 +/- 15%; 6-phospho-beta-galactosidase, 45,000. Enzyme II is a membrane-bound protein.     

Bacterial phosphoenolpyruvate-dependent phosphotransferase system: P-Ser-HPr and its possible regulatory function?

HPr of the bacterial phosphotransferase system is a histidine-containing phospho-carrier protein. It is phosphorylated at a single histidyl residue with phosphoenolpyruvate (PEP) and enzyme I and transfers the histidyl-bound phosphoryl group to a variety of factor III proteins. Recently, we described an HPr phosphorylated at a seryl residue (P-Ser-HPr), which is formed in an adenosine 5'-triphosphate dependent reaction catalyzed by a protein kinase [Deutscher, J., & Saier, M.-H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. Now we demonstrate that this P-Ser-HPr is an altered substrate of phosphorylated enzyme I and factor III proteins compared to unphosphorylated HPr. Thus, P-Ser-HPr of Streptococcus lactis is phosphorylated about 5000 times slower by PEP and enzyme I than HPr. The slow phosphorylation by PEP and enzyme I can be overcome when factor III protein specific for gluconate (factor III(Gct)) of Streptococcus faecalis is added. Most likely, a complex of P-Ser-HPr and factor III(Gct) is formed which then becomes phosphorylated as fast as free HPr. Factor III protein specific for lactose (factor III(Lac)) of Staphylococcus aureus also enhances the phosphorylation of P-Ser-HPr by enzyme I and PEP, but its effect is lower. Thus, P-Ser-HPr is phosphorylated 70-100-fold slower in the presence of factor III(Lac) than in the presence of factor III(Gct). The described interaction of P-Ser-HPr with enzyme I in the presence of different factor III proteins could account for the regulation of sugar uptake within the phosphotransferase system. Some of the phosphoenolpyruvate-dependent phosphotransferase system sugars like glucose are known to be taken up in preference to others, for example, lactose.     

Staphylococcal lactose phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis on the lacE gene gives evidence that a cysteine residue is responsible for phosphorylation

The lactose-specific phosphocarrier protein enzyme II of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacE gene in order to replace the histidine residues 245, 274 and 510 and the cysteine residue 476 of the amino acid sequence with a serine residue. The wild-type and mutant genes were expressed in Escherichia coli and the gene products were characterized in different in vitro test systems. In vitro phosphorylation studies on mutant derivatives of the lactose-specific enzyme II led to the conclusion that cysteine residue 476 is the active-site for phosphorylation of this enzyme II by a phospho-enzyme III of the same sugar specificity. A cysteine residue phosphorylated intermediate was first postulated for the mannitol-specific enzyme II of E. coli and studies performed independently concerning the lactose-specific enzyme II of Lactobacillus casei are in agreement with the above results.     

Enzyme IIIlac of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis of histidine residues, biochemical characterization and 1H-NMR studies

The lactose-specific phosphocarrier protein enzyme III of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacF gene in order to replace the histidine residues 78 and 82 of the amino acid sequence with a serine residue. Wild-type and both mutant genes were overexpressed in Escherichia coli and the gene products were purified to homogeneity. The conformation of wild-type and mutant proteins were monitored by 1H-NMR spectroscopy. In vitro phosphorylation studies on mutant lactose-specific enzyme III, as well as evidence from NMR spectroscopy, lead to the conclusion that His78 is the active-site for phosphorylation of lactose-specific enzyme III by phospho-HPr (histidine-containing protein). The role of His82 probably is the enhancement of velocity and efficiency of the phosphotransfer from lactose-specific enzyme III to lactose-specific enzyme II. This result refutes the conclusion of former work based on data by protelytic cleavage and sequencing of the 32P-labeled peptide of lactose-specific enzyme III that His82 is the active-site for phosphorylation.     

Amino-terminal sequences of the L, M, and H subunits of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

We have determined the sequence of the 25-28 amino-terminal residues of the three subunits, L, M, and H, of the membrane-bound reaction center protein of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. The sequences are as follows: L, H2N-Ala-Leu-Leu-Ser-Phe-Glu-Arg-Lys-Tyr-Arg- Val-Pro-Gly-Gly-Thr-Leu-Val-Gly-Gly-Asn-Leu-Phe-Asp-Phe-(His)-Val-; M, H2N-Ala-Glu-Tyr-Gln-Asn-Ile-Phe-Ser-Gln-Val-Gln-Val-Arg-Gly-Pro-Ala-Asp-Leu-Gly-Met-Thr-Glu-Asp-Val-Asn-Leu-Ala-Asn-; H, H2N-Met-Val-Gly-Val-Thr-Ala-Phe-Gly-Asn-Phe-Asp-Leu-Ala-Ser-Leu-Ala-Ile-Tyr-Ser-Phe-Trp-Ile-Phe-Leu-Ala-X-Leu-Ile-. The H sequence, especially after the aspartyl residue at position 11, is rich in hydrophobic residues, consistent with the possibility that this section of the polypeptide chain is located within the membrane. The L sequence is hydrophilic near the amino terminus and then becomes moderately hydrophobic. The M sequence is of average polarity.     

Biochemical characteristics of bovine retina nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.     

Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: purification and characterization of the phosphocarrier protein.

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble enzyme I, and a soluble phosphocarrier protein, HPr. The HPr has been purified to homogeneity by a combination of ammonium sulfate precipitations, gel filtration and diethylaminoethyl, carboxymethyl Bio-Gel A, and hydroxylapatite column chromatography. The purified protein is relatively heat stable (ca. 50% activity survives 30 min of boiling) and has a molecular weight of ca. 10,000 (determined by sodium dodecyl sulfate-gel electrophoresis and amino acid analysis). It contains a single histidine residue per molecule and can be totally inactivated by photooxidation with Rose Bengal dye. Although the mycoplasma HPr is very similar to that of Escherichia coli, it shows no significant association with antiserum produced against E. coli HPr.     

The primary structure of the subunits of carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum.

CO dehydrogenase/acetyl-coenzyme A synthase (CODH) is the central enzyme in the pathway of acetyl-coenzyme A biosynthesis in Clostridium thermoaceticum. It catalyzes the interconversion of CO and CO2 and the synthesis of acetyl-coenzyme A from the methylated corrinoid/iron sulfur protein, CO, and coenzyme A. It is a nickel-iron-sulfur protein and contains two subunits in the form (alpha beta)3. Reported here is the cloning and sequencing of the genes for both subunits of CODH. The gene for the alpha subunit codes for a protein with 729 amino acids and a molecular weight of 81,730, and the beta gene for a protein with 674 amino acids and a molecular weight of 72,928. The alpha subunit follows the beta subunit by 23 bases and the genes for both subunits are preceded by a sequence which is similar to the Shine-Dalgarno sequence of Escherichia coli. No significant amino acid sequence homology has been found to any known sequence. Labeling CODH with 2,4-dinitrophenylsulfenyl chloride and isolating labeled peptide fragments demonstrated that a tryptophan, residue 418 of the alpha subunit, is protected by coenzyme A and thus may be considered a potential part of the coenzyme A site.     

Relationship between mitochondrial NADH-ubiquinone reductase and a bacterial NAD-reducing hydrogenase

Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme. The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins. The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters. Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction. The mature protein is 444 amino acids long. Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16. This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN. The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal. The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN. Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3. Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits. This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.     

The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.

The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.     

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrose uptake-encoding region

The nucleotide sequence of the Vibrio alginolyticus sucrose uptake-encoding region was determined, and contained two genes, scrA and scrK. The scrA gene encodes an enzyme IISucrose (EIIScr) protein of the phosphoenolpyruvate dependent phosphotransferase system and the scrK gene encodes a fructokinase. The deduced amino acid (aa) sequence for the V. alginolyticus EIIScr protein was homologous with the EIIScr proteins from Streptococcus mutans, Salmonella typhimurium (pUR400 system) and Bacillus subtilis. The deduced aa sequence for the V. alginolyticus fructokinase was homologous with the Escherichia coli enzymes, 6-phosphofructokinase (isoenzyme 2) and ribokinase. Transposon phoA mutagenesis experiments indicated that the EIIScr protein was a membrane-bound protein with a region that extended into the periplasm.     

Sequences of 20 subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Application of a novel strategy for sequencing proteins using the polymerase chain reaction.

NADH:ubiquinone oxidoreductase, the first enzyme in the respiratory electron transport chain of mitochondria, is a membrane-bound multi-subunit assembly, and the bovine heart enzyme is now known to contain about 40 different polypeptides. Seven of them are encoded in the mitochondrial DNA; the remainder are the products of nuclear genes and are imported into the organelle. The primary structures of 12 of the nuclear coded subunits have been described and those of a further 20 are described here. The subunits have been sequenced by following a strategy based on the polymerase chain reaction. This strategy has been tailored from existing methods with the twofold aim of avoiding the use of cDNA libraries, and of obtaining a cDNA sequence rapidly with minimal knowledge of protein sequence, such as can be determined in a single N-terminal sequence experiment on a polypeptide spot on a two-dimensional gel. The utility and speed of this strategy have been demonstrated by sequencing cDNAs encoding 32 nuclear-coded-membrane associated proteins found in bovine heart mitochondria, and the procedures employed are illustrated with reference to the cDNA sequence of the 20 subunits of NADH:ubiquinone oxidoreductase that are presented. Extensive use has also been made of electrospray mass spectrometry to measure molecular masses of the purified subunits. This has corroborated the protein sequences of subunits with unmodified N terminals, and their measured molecular masses agree closely with those calculated from the protein sequences. Nine of the subunits, B8, B9, B12, B13, B14, B15, B17, B18 and B22 have modified alpha-amino groups. The measured molecular masses of subunits B8, B13, B14 and B17 are consistent with the post-translational removal of the initiator methionine and N-acetylation of the adjacent amino acid. The initiator methionine of subunit B18 has been removed and the N-terminal glycine modified by myristoylation. Subunits B9 and B12 appear to have N-terminal and other modifications of a hitherto unknown nature. The sequences of the subunits of bovine complex I provide important clues about the location of iron-sulphur clusters and substrate and cofactor binding sites, and give valuable information about the topology of the complex. No function has been ascribed to many of the subunits, but some of the sequences indicate the presence of hitherto unsuspected biochemical functions. Most notably the identification of an acyl carrier protein in both the bovine and Neurospora crassa complexes provides evidence that part of the complex may play a role in fatty acid biosynthesis in the organelle, possibly in the formation of cardiolipin.(ABSTRACT TRUNCATED AT 400 WORDS)     

A conserved proline in the last transmembrane segment of Gaa1 is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid. The nature of the functional interaction of the GPIT subunits with each other and with the proprotein and GPI substrates is largely unknown. We recently analyzed the GPIT subunit Gaa1, a polytopic protein with seven transmembrane (TM) spans, to identify sequence determinants in the protein that are required for its interaction with other subunits and for function (Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542). We showed that elimination of the C-terminal TM segment of Gaa1 allows the protein to interact with Gpi8, PIG-S, and PIG-T but renders the resulting GPIT complex nonfunctional. We now show that GPIT complexes containing C-terminally truncated Gaa1 possess a full complement of subunits and are able to interact with a proprotein substrate but cannot co-immunoprecipitate GPI. We go on to show that mutation of a conserved proline residue centrally located within the C-terminal TM span of Gaa1 is sufficient to abrogate the ability of the resulting GPIT complex to co-immunoprecipitate GPI. We suggest that the putative dynamic hinge created by the proline residue provides a structural basis for the interaction of GPI with GPIT.     

Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei. Evidence that a cysteine residue is essential for sugar phosphorylation

The gene coding for the lactose-specific Enzyme II of the Lactobacillus casei phosphoenolpyruvate-dependent phosphotransferase system, lacE, has been isolated by molecular cloning and expressed in Escherichia coli. The DNA sequence of the lacE gene and the deduced amino acid sequence are presented. The putative translation product comprises a hydrophobic protein of 577 amino acids with a calculated molecular mass of 62,350 Da. The deduced polypeptide has a high degree of sequence similarity with the corresponding lactose-specific enzymes II of Staphylococcus aureus and Lactococcus lactis. The sequence surrounding cysteine 483 was strongly conserved in the three proteins. The identity of the lacE product as the Enzyme IIlacL.casei was demonstrated by in vitro lactose phosphorylation assays using the protein expressed in E. coli. Single replacement of each of the histidine and cysteine residues by site-directed mutagenesis pointed to cysteine 483 as an amino acid residue essential for the phosphoryl group transfer reaction.     

Solution structure and dynamics of LuxU from Vibrio harveyi, a phosphotransferase protein involved in bacterial quorum sensing

The marine bacterium Vibrio harveyi controls its bioluminescence by a process known as quorum sensing. In this process, autoinducer molecules are detected by membrane-bound sensor kinase/response regulator proteins (LuxN and LuxQ) that relay a signal via a series of protein phosphorylation reactions to another response regulator protein, LuxO. Phosphorylated LuxO indirectly represses the expression of the proteins responsible for bioluminescence. Integral to this quorum sensing process is the function of the phosphotransferase protein, LuxU. LuxU acts to shuttle the phosphate from the membrane-bound proteins, LuxN and LuxQ, to LuxO. LuxU is a 114 amino acid residue monomeric protein. Solution NMR was used to determine the three-dimensional structure of LuxU. LuxU contains a four-helix bundle topology with the active-site histidine residue (His58) located on alpha-helix C and exposed to solution. The active site represents a cluster of positively charged residues located on an otherwise hydrophobic protein face. NMR spin-relaxation experiments identify a collection of flexible residues localized on the same region of LuxU as His58. The studies described here represent the first structural characterization of an isolated, monomeric bacterial phosphotransferase protein.     

Cleavage by protease from Staphylococcus aureus V8: an improvement in the sequence analysis of human hemoglobin variants

Protease from Staphylococcus aureus V8 cleaves either at glutamic residues or at both aspartic and glutamic residues, depending on the experimental conditions. In structural analyses of human hemoglobin variants, the specificity of this enzyme is of considerable interest to localize substitutions occurring in medium or large size peptides as it cleaves in smaller fragments which may be unambiguously characterized. It may also recognize the replacement of an acidic residue by the corresponding amide, or vice versa, avoiding protein sequence analysis. The various aspects of the use of protease V8 are illustrated by the study of four alpha chain hemoglobin variants concerning peptides alpha T-9 and alpha T-12b.     

Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli

Nitrate reductase, released and purified from membrane fractions of Escherichia coli, is composed of three subunits. Formation of the enzyme depends on induction of the nar operon, narGHJI, which is composed of four open reading frames (ORF). Previous studies established that the first two genes in the operon narG and narH encode the alpha and beta subunits, respectively, while formation of the gamma subunit, cytochrome bNR, depends on expression of the promoter distal genes. The narJ and narI genes were subcloned separately into plasmids where each was under the control of the nar promoter. Expression of these plasmids in a mutant which forms only alpha and beta subunits revealed that expression of the narI gene is sufficient to restore normal levels of cytochrome bNR, but expression of both genes is required for assembly of fully active, membrane-bound nitrate reductase. The amino acid composition, the N-terminal sequence, and the sequence of cyanogen bromide fragments derived from the isolated gamma subunit corresponds to that expected for a protein produced by the narI ORF. A protein corresponding to the narJ ORF did not appear to be associated with the purified nitrate reductase complex or with the complex immunoprecipitated from Triton X-100-solubilized membrane preparations. We conclude that narI encodes the gamma subunit (cytochrome bNR) and that while the product of the narJ gene is required for assembly of fully active membrane-bound enzyme it is not tightly associated with the active enzyme.