Biomedical subjects

L Hederstedt

Publications and source records attributed to L Hederstedt.

At least 55 records · Page 3Linked to original sources

PubMed1992

Organization and regulation of the Bacillus subtilis odhAB operon, which encodes two of the subenzymes of the 2-oxoglutarate dehydrogenase complex.

The primary structure of Bacillus subtilis 105 kDa 2-oxoglutarate dehydrogenase (E10) was deduced from the nucleotide sequence of the odhA gene and confirmed by N-terminal sequence analysis. The protein is highly homologous to E1o of Azotobacter vinelandii and Escherichia coli and of bakers' yeast cells. The 5' end of the odhAB mRNA was determined and the promoter region for the odhAB operon was localized to a 375 bp DNA fragment. The cellular concentration of the 4.5 kb odhAB transcript was found to be growth stage dependent; its concentration during growth in nutrient sporulation medium decreased abruptly at the end of the exponential growth phase and it was not detectable in early stationary phase. This decrease in the cellular concentration of the transcript is not the result of an increased rate of decay of the full-length odhAB mRNA, suggesting that transcription is down-regulated at the end of the exponential growth phase. The cellular concentration of the odhA and odhB gene products, E1o and dihydrolipoamide transsuccinylase (E2o), remains essentially constant throughout the growth curve in nutrient sporulation medium, indicating that both are rather stable proteins. In exponentially growing cells, glucose in nutrient sporulation medium repressed the cellular concentration of the odhAB mRNA, as well as that of E1o and E2o, about four-fold. This effect is most likely the result of a decreased rate of transcription from the odhAB promoter, since neither the stability nor the 5'-end of the transcript were affected by glucose in the medium. It is concluded that the cellular concentration of the 2-oxoglutarate dehydrogenase multienzyme complex (E1o and E2o) is regulated mainly at the transcriptional level.

Amino Acid Sequence↗

PubMed1992

Cloning and characterization of the Bacillus subtilis hemEHY gene cluster, which encodes protoheme IX biosynthetic enzymes.

Mutations that cause a block in a late step of the protoheme IX biosynthetic pathway, i.e., in a step after uroporphyrinogen III, map at 94 degrees on the Bacillus subtilis chromosomal genetic map. We have cloned and sequenced the hem genes at this location. The sequenced region contains six open reading frames: ponA, hemE, hemH, hemY, ORFA, and ORFB. The ponA gene product shows over 30% sequence identity to penicillin-binding proteins 1A of Escherichia coli, Streptococcus pneumoniae, and Streptococcus oralis and probably has a role in cell wall metabolism. The hemE gene was identified from amino acid sequence comparisons as encoding uroporphyrinogen III decarboxylase. The hemH gene was identified by enzyme activity analysis of the HemH protein expressed in E. coli. It encodes a water-soluble ferrochelatase which catalyzes the final step in protoheme IX synthesis, the insertion of ferrous iron into protoporphyrin IX. The function of the hemY gene product was not elucidated, but mutation analysis shows that it is required for a late step in protoheme IX synthesis. The hemY gene probably encodes an enzyme with coproporphyrinogen III oxidase or protoporphyrinogen IX oxidase activity or both of these activities. Inactivation of the ORFA and ORFB genes did not block protoheme IX synthesis. Preliminary evidence for a hemEHY mRNA was obtained, and a promoter region located in front of hemE was identified. From these combined results we conclude that the hemEHY gene cluster encodes enzymes for the synthesis of protoheme IX from uroporphyrinogen III and probably constitutes an operon.

Amino Acid Sequence↗

PubMed1991

Bacillus subtilis cytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa3-type oxidases.

The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127 degrees on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N',N'-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from ctaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify subunit II, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.

Aspartic Acid↗

PubMed1991

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III.

We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

Amino Acid Sequence↗

PubMed1990

Low temperature EPR and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate: quinone oxidoreductase indicate bis-histidine coordination of the heme iron.

Bacillus subtilis cytochrome b-558 was expressed in high amounts in Escherichia coli, solubilized from membranes with detergent and purified free from other hemoproteins. The cytochrome possibly contains two heme groups. To determine the axial ligands to the low-spin heme and the heme rhombicity, the cytochrome was analyzed using low-temperature electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopy. The combined results exclude bis-methionine, bis-lysine and histidine-methionine coordination. Bis-histidine coordination of the heme(s) with a near perpendicular orientation of the imidazole planes is strongly suggested by the highly axial low-spin EPR signals and the intense near infrared MCD spectrum (delta epsilon = 380 M-1.cm-1 at 4.2 K and 5 T) of the charge-transfer band at 1600 nm.

Bacillus subtilis↗

PubMed1990

Bacillus subtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli.

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous cytochrome c and suggests that cytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis cytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-terminal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.

Alkaline Phosphatase↗

PubMed1990

Bacillus subtilis 13-kilodalton cytochrome c-550 encoded by cccA consists of a membrane-anchor and a heme domain.

Little is known about c-type cytochromes in Gram-positive bacteria in contrast to the wealth of information available on this type of cytochrome in Gram-negative bacteria and in eucaryotes. In the present work, the strictly aerobic bacterium Bacillus subtilis was analyzed for subcellular localization and number of different cytochromes c. In vivo labeling with radioactive 5-aminolevulinic acid, a precursor to heme, showed that the proteins containing covalently bound heme are predominantly found in the membrane fraction. One major membrane-bound cytochrome c of about 15 kDa and with an alpha-band absorption peak in the reduced state at 550 nm was analyzed in more detail. Cytochrome c-550 has the properties of an integral membrane protein. The physiological function of this relatively high redox potential cytochrome is not known. Its structural gene, cccA, was cloned, sequenced, and overexpressed in B. subtilis. The gene maps adjacent to rpoD (sigA) at 223 degrees on the chromosome. The amino acid sequence of cytochrome c-550 as deduced from the DNA sequence consists of 120 residues and contains one heme c binding site (Cys-Ile-Ala-Cys-His) located approximately in the middle of the polypeptide. From the hydropathy distribution and from comparisons to soluble c-type cytochromes of known three-dimensional structure, cytochrome c-550 seemingly consists of two domains; an N-terminal membrane-anchor domain and a C-terminal heme domain. A model for the topography of the cytochrome in the cytoplasmic membrane is suggested in which the N-terminal part spans the membrane in the form of a single segment in an alpha-helical conformation and the C-terminal heme domain is exposed on the extracytoplasmic side of the membrane. Deletion of cccA from the chromosome revealed another membrane-bound cytochrome with absorption maximum at 550 nm in the reduced state. Analysis of cccA deletion mutants demonstrated that the cytochrome c-550 encoded by cccA is not essential for growth of B. subtilis on rich or minimal media.

Amino Acid Sequence↗

PubMed1990

Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II).

Cytochrome b558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron-sulphur protein (Ip) in succinate:quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis-imidazole ligation of the haem. Apo-cytochrome was found in the membrane fraction of haem-deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His-47 can be excluded as a haem ligand. The remaining His residues (at positions 13, 28, 70, 113 and 155) are located in or close to four predicted transmembrane segments. The Tyr-28 and Tyr-70 mutant proteins appeared to lack one of the two haems. Only the Tyr-13 and Tyr-47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr-13 mutant cytochrome assembles into an enzymatically defective succinate:quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His-28, His-70 and His-155 are probably haem axial ligands in a dihaem cytochrome b558. His-70 and His-155 may be ligands to the same haem.

Amino Acid Sequence↗

PubMed1990

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.

Aldehyde Oxidoreductases↗

PubMed1989

EPR characterization of soluble fragments of succinate dehydrogenase from mutant strains of Bacillus subtilis.

Succinate dehydrogenase is a membrane-bound metallo-flavo-enzyme containing a bi- (S-1), a tri- (S-3) and a tetranuclear (S-2) iron-sulfur cluster. The catalytic portion of the enzyme contains two distinct subunits designated Fp and Ip. Using concentrated extracts from mutant strains of Bacillus subtilis it was demonstrated, by using low temperature EPR, that cluster S-2 can be assembled in a soluble succinate dehydrogenase. In a mutant with a truncated Ip subunit which lacks 7 of the 11 conserved cysteine residues, cluster S-1 lacked the spin relaxation properties attributable to an adjacent cluster S-2. These data are consistent with a model where one or more cysteine residues from the middle set of 4 conserved cysteines in the Ip subunit are ligands to the tetranuclear cluster.

Bacillus subtilis↗

PubMed1989

The structural gene for aspartokinase II in Bacillus subtilis is closely linked to the sdh operon.

The aecA and aecB loci map at 250 and 290 degrees, respectively, on the Bacillus subtilis chromosomal genetic map. The aecB locus has been proposed as the structural gene for aspartokinase II. From DNA sequence analyses and comparisons to the sequence of the aspartokinase II gene, it can be concluded that the structural gene for aspartokinase II is located close to sdh at 250 degrees and cannot be aecB. A detailed map over 7 kbp in the 250 degree region is presented.

Aspartate Kinase↗

PubMed1989

New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation.

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.

Bacillus subtilis↗

PubMed1989

Genetic characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively.

The 2-oxoglutarate dehydrogenase complex consists of three different subenzymes, the E1o (2-oxoglutarate dehydrogenase) component, the E2o (dihydrolipoyl transsuccinylase) component, and the E3 (dihydrolipoamide dehydrogenase) component. In Bacillus subtilis, the E1o and E2o subenzymes are encoded by odhA and odhB, respectively. A plasmid with a 6.8-kilobase-pair DNA fragment containing odhA and odhB was isolated. Functional E1o and E2o are expressed from this plasmid in Escherichia coli. Antisera generated against B. subtilis E1o and E2o expressed in E. coli reacted with antigens of the same size from B. subtilis. The nucleotide sequence of odhB and the terminal part of odhA was determined. The deduced primary sequence of B. subtilis E2o shows striking similarity to the corresponding E. coli protein, which made it possible to identify the lipoyl-binding lysine residue as well as catalytic histidine and aspartic acid residues. An mRNA of 4.5 kilobases hybridizing to both odhA and odhB probes was detected, indicating that odhA and odhB form an operon.

Acyltransferases↗

PubMed1988

Ligands to the 2Fe iron-sulfur center in succinate dehydrogenase.

Membrane-bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron-sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtilis succinate dehydrogenase-defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe-2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N-terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin-containing subunit of the dehydrogenase.

Bacillus subtilis↗

PubMed1987

Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558.

Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His-113 excludes this residue as an axial heme ligand. A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B. subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.

Bacillus subtilis↗

PubMed1987

Processing of Bacillus subtilis succinate dehydrogenase and cytochrome b-558 polypeptides. Lack of covalently bound flavin in the Bacillus enzyme expressed in Escherichia coli.

The DNA sequence of the Bacillus subtilis sdh operon coding for the two succinate dehydrogenase subunits and cytochrome b-558 (the membrane anchor protein) has recently been established. We have now determined the extent of N-terminal processing of each polypeptide by radiosequence analysis. At the same time, direct evidence for the correctness of the predicted reading frames has been obtained. The cytochrome showed a ragged N-terminus, with forms lacking one residue, and is inserted across the membrane without an N-terminal leader-peptide. Covalently bound flavin was not detectable in B. subtilis succinate dehydrogenase expressed in Escherichia coli despite normal N-terminal processing of the apoprotein. This provides an explanation to why the succinate dehydrogenase synthesized in E. coli is not functional and demonstrates that host-specific factors regulate the coenzyme attachment.

Amino Acid Sequence↗

PubMed1987

Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli.

The 2-oxoglutarate dehydrogenase multienzyme complex is composed of three different subenzymes: 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide transsuccinylase (E2o), and dihydrolipoamide dehydrogenase (E3). Bacillus subtilis E1o and E2o are encoded by the citK and citM genes, respectively. A 3.4-kb BamHI DNA fragment containing citK and citM markers was isolated from a library of B. subtilis DNA in Escherichia coli. Functional E2o was expressed from the cloned DNA both in B. subtilis and E. coli. E2o had an apparent Mr of 60,000 when expressed in E. coli. The B. subtilis E2o component complemented an E. coli E2o-defective mutant in vivo and in vitro. It is concluded that functional B. subtilis E2o can be produced in E. coli and can interact with E. coli and E1o and E3 to form an active chimeric enzyme complex.

Acyltransferases↗

PubMed1987

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.

Amino Acid Sequence↗