BpuAI, a novel BbsI and BbvII isoschizomer from Bacillus pumilus recognizing 5'-GAAGAC-3'.
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Bacillus↗
Biomedical subjects
W Hengstenberg
Publications and source records attributed to W Hengstenberg.
Mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus. Sequence and expression in Escherichia coli and structural comparison with the enzyme IImannitol of Escherichia coli.
The enzyme IImannitol (EIImtl) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the uptake and concomitant phosphorylation of mannitol by bacteria; it is specified by the gene mtlA. MtlA is located near the genes mtlF and mtlD in the staphylococcal genome, encoding the enzyme IIImtl and the mannitol-1-phosphate dehydrogenase, respectively. We present the cloning of the whole operon by a novel complementation system which is generally suitable for cloning Gram-positive PTS genes. The nucleotide sequence of a 2.5-kbp subclone spanning mtlA has been determined. From the deduced amino acid sequence, it is predicted that the membrane-protein EIImtl consists of 505 amino acid residues (54112 Da). The protein has the expected hydropathy profile of an integral-membrane protein. The NH2-terminal part of the enzyme resides within the membrane, whereas the COOH-terminus of the enzyme has the properties of a soluble protein. Comparison with the known amino acid sequence of EIImtl of Escherichia coli [Lee, C. A. & Saier, M. H. (1983) J. Biol. Chem. 258, 10761-10767] showed significant similarity. The motif containing the cysteine, which is the putative second phosphorylation site in EIImtl of E. coli [Pas, H. H. & Robillard, G. T. (1988) Biochemistry 27, 5835-5839], is well conserved in EIImtl of Staphylococcus carnosus. Chemical modification of the single active site cysteine residue by Ellman's reagent leads to total inactivation, which can be reversed by treatment with 2-mercaptoethanol.
Amino Acid Sequence↗
A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-beta-galactosidase.
The lacG gene encoding the 6-phospho-beta-galactosidase (E.C.3.2.1.85) of Staphylococcus aureus was fused to the protein A gene in the plasmid pRIT2T. Escherichia coli cells containing this plasmid produce a fusion protein with both IgG binding and 6-phospho-beta-galactosidase activities after heat induction. The recombinant gene was overexpressed and the hybrid protein was purified to homogeneity in high yield. The chimeric protein was shown to have almost identical enzymatic characteristics to pure 6-phospho-beta-galactosidase. This result leads to the conclusion that a free N-terminus of the 6-phospho-beta-galactosidase is not required for biological activity. The hybrid protein of protein A and 6-phospho-beta-galactosidase was used as an enzyme conjugate in enzyme-linked immunosorbent assays (ELISA). The experiments presented demonstrate that the 6-phospho-beta-galactosidase is a suitable fusion partner in various diagnostic applications where an unique biological activity is required.
Amino Acid Sequence↗
Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: molecular cloning and nucleotide sequence of the Staphylococcus carnosus ptsI gene and expression and complementation studies of the gene product.
A digoxigenin-labeled DNA probe that was complementary to the gene ptsH and the beginning of the gene ptsI was used to clone a 3.2-kb HincII-BamHI restriction fragment containing the complete ptsI gene of Staphylococcus carnosus. The restriction fragment was cloned in the antisense orientation to the lac promoter in the low-copy-number vector pSU18. The nucleotide sequences of the ptsI gene, which encodes enzyme I (EC 2.7.3.9), and the corresponding flanking regions were determined. The primary translation product, derived from the nucleotide sequence, consists of 574 amino acids and has a calculated molecular weight of 63,369. Amino acid sequence comparison showed 47% similarity to enzyme I of Escherichia coli and 37% similarity to the enzyme I domain of the multiphosphoryl transfer protein of Rhodobacter capsulatus. The histidinyl residue at position 191 could be identified as the probable phosphoenolpyruvate-dependent phosphorylation site of enzyme I of S. carnosus because of sequence homologies with the peptide sequences of enzyme I-active sites of Enterococcus faecalis and Lactococcus lactis. Several in vivo and in vitro complementation studies with the enzyme I ptsI genes of S. carnosus and the E. coli ptsI mutant JLT2 were carried out. The generation times and interaction between enzyme I with histidine-containing protein from gram-positive and gram-negative bacteria were measured in a phosphoryl group transfer test.
Amino Acid Sequence↗
Two-dimensional 1H NMR studies on HPr protein from Staphylococcus aureus: complete sequential assignments and secondary structure.
Complete sequence-specific assignments of the 1H NMR spectrum of HPr protein from Staphylococcus aureus were obtained by two-dimensional NMR methods. Important secondary structure elements that can be derived from the observed nuclear Overhauser effects are a large antiparallel beta-pleated sheet consisting of four strands, A, B, C, D, a segment SAB consisting of an extended region around the active-center histidine (His-15) and an alpha-helix, a half-turn between strands B and C, a segment SCD which shows no typical secondary structure, and the alpha-helical, C-terminal segment S(term). These general structural features are similar to those found earlier in HPr proteins from different microorganisms such as Escherichia coli, Bacillus subtilis, and Streptococcus faecalis.
Amino Acid Sequence↗
Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and protein sequencing of the Staphylococcus carnosus histidine-containing protein, and cloning and DNA sequencing of the ptsH gene.
The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr-expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy-chain-termination technique. Upstream, the 264-bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.
Amino Acid Sequence↗
Exchange of phosphoryl groups between HPr molecules of the phosphoenolpyruvate-dependent phosphotransferase system is an autocatalytic process.
HPr, a central component of the phosphoenolpyruvate-dependent phosphotransferase system, can exist in Escherichia coli in a phosphorylated (PHPr) and a nonphosphorylated form. We show that, beside the normal transfer of the phosphoryl group from PHPr to enzymes II and III, PHPr can phosphorylate other HPr molecules in an autocatalytic exchange reaction. The reaction is very fast but is inhibited by labeling the protein with Bolton-Hunter reagent. We demonstrate that the exchange reaction can be used to determine the delta G degree of the phosphoryl group of mutant forms of PHPr relative to wild-type PHPr. Two HPr mutants were constructed by site-directed mutagenesis, HPr P11E and HPr E68A. Both show altered phosphoryl group potentials but show no significantly altered KM or Vmax values compared to wild-type HPr, illustrating the sensitivity of the exchange process. The exchange reaction does not occur between HPr from E. coli and HPr from Staphylococcus carnosus.
Autoradiography↗
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.
Bacterial Proteins↗
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.
Amino Acid Sequence↗
Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes.
Enzyme IIIMtl is part of the mannitol phosphotransferase system of Enterococcus faecalis. It is phosphorylated in a reaction sequence requiring enzyme I and heat-stable phosphocarrier protein (HPr). The phospho group is transferred from enzyme IIIMtl to enzyme IIMtl, which then catalyzes the uptake and concomitant phosphorylation of mannitol. The internalized mannitol-1-phosphate is oxidized to fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. In this report we describe the cloning of the mtlF and mtlD genes, encoding enzyme IIIMtl and mannitol-1-phosphate dehydrogenase of E. faecalis, by a complementation system designed for cloning of gram-positive phosphotransferase system genes. The complete nucleotide sequences of mtlF, mtlD, and flanking regions were determined. From the gene sequences, the primary translation products are deduced to consist of 145 amino acids (enzyme IIIMtl) and 374 amino acids (mannitol-1-phosphate dehydrogenase). Amino acid sequence comparison confirmed a 41% similarity of E. faecalis enzyme IIIMtl to the hydrophilic enzyme IIIMtl-like portion of enzyme IIMtl of Escherichia coli and 45% similarity to enzyme IIIMtl of Staphylococcus carnosus. The putative N-terminal NAD+ binding domain of mannitol-1-phosphate dehydrogenase of E. faecalis shows a high degree of similarity with the N terminus of E. coli mannitol-1-phosphate dehydrogenase (T. Davis, M. Yamada, M. Elgort, and M. H. Saier, Jr., Mol. Microbiol. 2:405-412, 1988) and the N-terminal part of the translation product of S. carnosus mtlD, which was also determined in this study. There is 40% similarity between the dehydrogenases of E. faecalis and E. coli over the whole length of the enzymes. The organization of mannitol-specific genes in E. faecalis seems to be similar to the organization in S. carnosus. The open reading frame for enzyme IIIMtl E. faecalis is followed by a stem-loop structure, analogous to a typical Rho-independent terminator. We conclude that the mannitol-specific genes are organized in an operon and that the gene order is mtlA orfX mtlF mtlD.
Amino Acid Sequence↗
Studies for the elucidation of the mode of action of the antimycotic hydroxypyridone compound, rilopirox.
Rilopirox is a synthetic, fungicidal antimycotic agent with hydrophobic characteristics. Its chemical name is 6-[4-(4-chlorophenoxy)-phenoxy-methyl]-1-hydroxy-4-methyl-2-pyridone and it has a molecular weight of 357.79. Rilopirox is very soluble in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) but poorly soluble in water. The amount of antimycotic agent remaining in the solution is dependent on the final concentration of the solvent and the amount of rilopirox used. Complexometric studies show that rilopirox has a high affinity for iron ions [unpubl. data]. Catalase, an iron-containing enzyme, is inhibited by the chelating agent rilopirox. Studies on yeast mitochondria and submitochondrial particles show that rilopirox inhibits the respiratory chain. Complex I (NADH-ubiquinone oxidoreductase) contains iron-sulfur proteins and is the main system which is inhibited.
Antifungal Agents↗
Characterization of the lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis.
The plasmid-encoded lactose genes of the Lactococcus lactis phosphotransferase system encoding Enzyme IIIlac (lacF) and Enzyme IIlac (lacE) have been identified and cloned in Escherichia coli and L. lactis. Nucleotide sequence and transcription analysis showed that these genes are organized into a lactose-inducible operon with the gene order lacF-lacE-lacG-lacX, the latter two genes encoding phospho-beta-galactosidase and a 34-kDa protein with an unknown function, respectively. The lac-operon is immediately followed by an IS element that is homologous to ISS1. Enzyme IIIlac was purified from L. lactis and determination of its NH2-terminal sequence demonstrated that the lacF gene starts with a TTG codon and encodes a 105 amino acid protein (Mr = 11416). Cross-linking studies with the purified enzyme showed that Enzyme IIIlac is active as a trimer. A mutant lacF gene was identified in strain YP2-5 and appeared to encode Enzyme IIIlac containing the missense mutation G18E. The lacF gene could be expressed under control of vector-located promoter sequences resulting in overproduction of Enzyme IIIlac in E. coli and complementation of the L. lactis lacF mutant YP2-5. The deduced amino acid sequence of Enzyme IIlac consists of 586 amino acids (Mr = 61562) and shows the characteristics of a hydrophobic, integral membrane protein. The deduced primary structures of the L. lactis Enzyme IIIlac and Enzyme IIlac are homologous to those of Staphylococcus aureus (72 and 71% identity, respectively) and Lactobacillus casei (48 and 47% identity, respectively). In contrast, the organization of the lactose genes differs significantly between those Gram-positive bacteria. Heterogramic homology in specific domains was observed between the derived amino acid sequences of the lactose-specific enzymes and that of E. coli Enzyme IIIcel and Enzyme IIcel, which suggest a common function in the transport and phosphorylation of these structurally related beta-glucosides.
Amino Acid Sequence↗
The nucleotide sequence of ptsH gene from Klebsiella pneumoniae.
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Amino Acid Sequence↗
Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus.
The gene which encodes the mannitol-specific enzyme III (EIIImtl) of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus, has been cloned. Genomic libraries of S. carnosus DNA were constructed using the expression vector pUC19 and EIIImtl-producing clones were identified using rabbit polyclonal antiserum. A 700-bp Dde I fragment, containing the complete gene encoding EIIImtl, was sequenced by the dideoxy chain-termination technique. Upstream from the ORF for EIIImtl one can find a sequence analogous to that of the Escherichia coli promoter. This region acts as a strong promoter when subcloned into the promoter test vector M13HDL17. EIIImtl was overproduced using the inducible T7 polymerase system and purified to homogeneity. Amino acid sequence comparison confirmed a 38% similarity to the hydrophilic enzyme-III-like portion of enzyme IImtl of E. coli. There is also a 36% similarity to the N terminus of the fructose-specific phospho-carrier protein from E. coli.
Amino Acid Sequence↗
Structure and function of proteins involved in sugar transport by the PTS of gram-positive bacteria.
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Amino Acid Sequence↗
Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site.
The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins. In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.
Adenosine Triphosphate↗
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.
Amino Acid Sequence↗
Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus faecalis.
The phosphocarrier protein HPr of the bacterial phosphoenolpyruvate:sugar phosphotransferase system contains 1-phosphohistidine at residue 15. This residue and the active site residue Arg-17 are conserved in HPrs isolated from both Gram-positive and -negative bacteria. The pH- and temperature-dependent hydrolysis of the 1-phosphohistidinyl residue in P-HPr from Streptococcus faecalis has been investigated. The results show that the hydrolysis properties are very similar to those previously reported for P-HPr from Escherichia coli. It was postulated that the unusual hydrolysis properties were due to the presence of a carboxyl group at the active site, and it is now known that in HPr from Escherichia coli the C-terminal residue Glu-85 is present. The results in this paper suggest that a similar carboxyl group is present at the active site in HPr from Streptococcus faecalis.
Amino Acid Sequence↗