Characterization of a structural model of membrane bound cytochrome c-550 from Bacillus subtilis

In order to identify inhibitors of various drug-resistant forms of the human immunodeficiency virus protease (HIV PR), we have designed and synthesized pseudopeptide libraries with a general structure Z-mimetic-Aa1-Aa2-NH2. Five different chemistries for peptide bond replacement have been employed and the resulting five individual sublibraries tested with the HIV PR and its drug-resistant mutants. Each mutant contains amino acid substitutions that have previously been shown to be associated with resistance to protease inhibitors, including Ritonavir, Indinavir, and Saquinavir. We have mapped the subsite preferences of resistant HIV PR species with the aim of selecting a pluripotent pharmaceutical lead. All of the enzyme species in this study manifest clear preference for an L-Glu residue in the P2' position. Slight, but significant, differences in P3' subsite specificity among individual resistant PR species have been documented. We have identified three compounds, combining the most favorable features of the inhibitor array, that exhibit low-nanomolar or picomolar Ki values for all three mutant PR species tested.     

Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis.

Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential 5' maturation of all precursor tRNA molecules. The protein component both alters the conformation of the RNA component and enhances the substrate affinity and specificity. To facilitate biochemical and biophysical studies, the protein component of Bacillus subtilis ribonuclease P (RNase P) was overproduced in Escherichia coli using the native amino acid sequence with the initial 20 codons optimized for expression in E.coli . A simple purification procedure using consecutive cation exchange chromatography steps in the presence and absence of urea was developed to purify large quantities of P protein without contaminating nucleic acids. The identity of the recombinant protein as a cofactor of RNase P was established by its ability to stimulate the activity of the RNA component in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism studies indicate that P protein is a combination of alpha-helix and beta-sheet secondary structures and is quite stable, with a T m of 67 degrees C. The described methods facilitated the large scale purification of homogeneous, RNA-free P protein required for high resolution crystallographic analyses and may be useful for the preparation of other RNA binding proteins.     

Characterization of cytochrome c-550 from cyanobacteria

Cytochrome c-550 has been purified from several cyanobacteria. It is a low-potential, auto-oxidizable cytochrome. This cytochrome should not be confused with a degradation product of cytochrome ? which may be formed during the isolation of the latter protein. Cytochromes c-550 are distinctive in size, amino-acid composition and N-terminal amino-acid sequence.     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 ℃ .     

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg-1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65℃ and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65℃.     

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.     

Expression, mutagenesis, and characterization of recombinant low-potential cytochrome c550 of photosystem II

Cytochrome c(550) of the photosystem II complex of cyanobacteria is an unusual member of the large protein family of monoheme c-type cytochromes. Despite the fact that it shares considerable amino acid sequence similarity and has a protein fold similar to the other members of the family, Cyt.c(550) has a midpoint potential (E(m7) = -250 mV) that is much lower than the positive midpoint potentials characteristic (E(m7) = 100-300mV) of this cytochrome family. An E. coli heterologous expression system involving secretion of the recombinant protein from Synechocystis PCC6803 to the periplasm was utilized to allow production of wild-type and mutant forms of the cytochrome. For most of the variants studied, the yield of protein was significantly enhanced by growth at 28 degrees C and inclusion of sucrose and betaine, in addition to isopropyl-beta-d-thiogalactoside (IPTG), to the growth medium of the E. coli expression host. Analysis of the protein products revealed that the wild-type protein maintained the redox and visible spectroscopic characteristics of the authentic protein. Mutations in the residues engaging in hydrogen bond interactions with the heme propionate (Asn49) and the axial 6th ligand His92 (Pro93) resulted in small (12-20 mV), but reproducible, upshifts in midpoint redox potential. Substitution of the axial ligand His92 with Met produced no discernible changes in the optical spectrum relative to the wild-type despite the fact that in this mutant, unlike the others studied here, the thioether linkage either was not formed or was highly labile as evidenced by loss of the heme during SDS-PAGE. On the other hand, the midpoint potential of the C550-H92M mutant was upshifted by approximately 70 mV. This value is significantly less of a perturbation than that observed in a similar mutant that is natively expressed in Thermosynechoccocus but appears to have an intact thioether linkage between the heme and the polypeptide moiety.     

Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein.

In DNA binding-deficient mutants of Bacillus subtilis a competence-specific protein with a subunit molecular weight of 18,000 was absent. The native protein containing this subunit was purified from B. subtilis membranes by chromatography on hydroxyapatite, DEAE-cellulose, and Sephacryl S-200. This protein appeared to be complexed with a second protein of slightly lower molecular weight (17,000) and a different isoelectric point. The native protein complex (apparent molecular weight, 75,000) contained approximately equal amounts of the two polypeptides and showed a strong DNA-binding activity. Incubation of the complex with plasmid and bacteriophage DNA revealed nuclease activity, specifically directed toward double-stranded DNA. Predominantly single-stranded nicks and a limited number of double-stranded breaks were introduced in the presence of Mg2+ ions. In the presence of Mn2+ ions the complex produced low-molecular-weight breakdown products from the DNA.     

Kinetic characterization of cytochrome c oxidase from Bacillus subtilis

Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.     

Expression, purification, and biochemical properties of arginase from Bacillus subtilis 168

The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The K_m and V_max values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.     

Expression, purification, and characterization of the CuA-cytochrome c domain from subunit II of the Bacillus subtilis cytochrome caa3 complex in Escherichia coli

Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.     

Expression, purification, and characterization of BsSco, an accessory protein involved in the assembly of cytochrome c oxidase in Bacillus subtilis

The studies described here were performed to characterize further the plasma membrane associated protein BsSco, which is the product of the gene ypmQ, in Bacillus subtilis. BsSco is a member of the Sco family of proteins found in the inner mitochondrial membrane of yeast and humans and implicated as an accessory protein in the assembly of the Cu(A) site of cytochrome c oxidase. We have cloned the gene expressing BsSco, placed a six-histidine tag on its C-terminus, and over-expressed this protein in B. subtilis. Recombinant BsSco with the his-tag has been purified from Triton X-100-solubilized plasma membranes by nickel metal affinity chromatography. Mass spectral analysis of the purified protein is consistent with processing of BsSco by signal peptidase II removing an N-terminal putative transmembrane sequence to leave an acyl-glyceryl moiety at cysteine residue 19. Antibodies, raised against purified, recombinant BsSco, were used to characterize the timing of the level of native BsSco in batch cultures of wild-type B. subtilis. There is a marked lag in the level of native BsSco, but it does appear prior to cytochrome c oxidase, which is expressed in late stage growth. This work supports a role for BsSco in the assembly of the Cu(A) site of cytochrome c oxidase and its functional relationship to the Sco proteins found in eukaryotic cells.     

Expression, purification, and characterization of BioI: a carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene bioI. We have subcloned biol and overexpressed the encoded protein, Biol. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography. Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coli acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis. In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production.     

Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.     

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.     

Catabolite regulation of the cytochrome c550-encoding Bacillus subtilis cccA gene

In Gram-positive bacteria, catabolite control protein A (CcpA)-mediated catabolite repression or activation regulates not only the expression of a great number of catabolic operons, but also the synthesis of enzymes of central metabolic pathways. We found that a constituent of the Bacillus subtilis respiratory chain, the small cytochrome c550 encoded by the cccA gene, was also submitted to catabolite repression. Similar to most catabolite-repressed genes and operons, the Bacillus subtilis cccA gene contains a potential catabolite response element cre, an operator site recognized by CcpA. The presumed cre overlaps the -35 region of the cccA promoter. Strains carrying a cccA'-IacZ fusion formed blue colonies when grown on rich solid medium, whereas white colonies were obtained when glucose was present. beta-Galactosidase assays with cells grown in rich medium confirmed the repressive effect of glucose on cccA'-lacZ expression. Introduction of a ccpA or hprK mutation or of a mutation affecting the presumed cccA cre relieved the repressive effect of glucose during late log phase. An additional glucose repression mechanism was activated during stationary phase, which was not relieved by the ccpA, hprK or cre mutations. An interaction of the repressor/corepressor complex (CcpA/seryl-phosphorylated HPr (P-Ser-HPr)) with the cccA cre could be demonstrated by gel shift experiments. By contrast, a DNA fragment carrying mutations in the presumed cccA cre was barely shifted by the CcpA/P-Ser-HPr complex. In footprinting experiments, the region corresponding to the presumed cccA cre was specifically protected in the presence of the CcpA/P-Ser-HPr complex.