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.     

Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2-oxoglutarate dehydrogenase complex.

Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2-oxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino-acid residues) and a mature protein (387 residues, Mr = 41 534). Recombinant porcine E2o (rPE2o) (residues 1-387), C- and N-terminal truncated PE2os, and site-directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET-11d and purified. The succinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube-like structure very similar to that of the native PE2o. Deletion of five amino-acid residues from the C-terminus resulted in a complete loss of both enzymatic activity and formation of the cube-like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C-terminal leucine triplet (Leu383-384-385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383-384-385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5-fold reduction in kcat, with little change in Km values for dihydrolipoamide and succinyl-CoA. However, self-assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383-384-385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o.     

Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.     

Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.

Sequences located in the N-terminal region of the high M(r) 2-oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N-terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1') and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This 'lipoyl-like' domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1' with trypsin both cause loss of overall OGDC function although the E1' fragment retains full catalytic activity. Removal of this small N-terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC.     

In vitro complementation of Bacillus subtilis and Escherichia coli. 2-oxoglutarate dehydrogenase complex mutants and genetic mapping of B. subtilis citK and citM mutations

Abstract The 2-oxoglutarate dehydrogenase complex of the tricarboxylic acid cycle (TCA) consists of multiple copies of 3 different subenzymes; E₁, E₂ and E₃. The E₃ subenzyme is also a component of the pyruvate dehydrogenase complex. Bacillus subtilis 2-oxoglutarate dehydrogenase mutants were studied. The mutants defective in E₁, E₂ and E₃ subenzyme activity, respectively, could be separated into 3 groups by biochemical complementation analyses. The groups correspond to the citK, citM and citL genes. A B. subtilis subenzyme defect, probably E₁, could be complemented with the corresponding Escherichia coli wild-type subenzyme and vice versa. Mutations in citK and citM are closely linked. The gene order kauA——citK-citM was determined from 3-factor transformation crosses. It is concluded that the gene organization and the subenzyme structure of the 2-oxoglutarate dehydrogenase complex are similar in B. subtilis and E. coli .     

Purification and characterization of NADH dehydrogenase from Bacillus subtilis

NADH dehydrogenase from Bacillus subtilis W23 has been isolated from membrane vesicles solubilized with 0.1% Triton X-100 by hydrophobic interaction chromatography on an octyl-Sepharose CL-4B column. A 70-fold purification is achieved. No other components could be detected with sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ferguson plots of the purified protein indicated no anomalous binding of sodium dodecyl sulphate and an accurate molecular weight of 63 000 could be determined. From the amino acid composition a polarity of 43.8% was calculated indicating that the protein is not very hydrophobic. Optical absorption spectra and acid extraction of the enzyme chromophore followed by thin-layer chromatography showed that the enzyme contains 1 molecule FAD/molecule. The enzyme was found to be specific for NADH. NADPH is oxidized at a rate which is less than 6% of the rate of NADH oxidation. The activity of the enzyme as determined by NADH:3-(4'-5'-dimethyl-thiazol-2-yl)2,4-diphenyltetrazolium bromide oxidoreduction is optimal at 37 C and pH 7.5-8.0. The purified enzyme has a Kapp for NADH of 60 microM and a V of 23.5 mumol NADH/min X mg protein. These parameters are not influenced by phospholipids. The enzyme activity is hardly or not at all affected by NADH-related compounds such as ATP, ADP, AMP, adenosine, deoxyadenosine, adenine and nicotinic amide indicating the high binding specificity of the enzyme for NADH.     

Structural and enzymatic characterization of Bacillus subtilis R,R-2,3-butanediol dehydrogenase

2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76) also known as acetoin reductase (AR, EC 1.1.1.4) is the key enzyme converting acetoin (AC) into 2,3-butanediol (BD) and undertaking the irreversible conversion of diacetyl to acetoin in various microorganisms. The existence of three BDHs (R,R-, meso-, and S,S-BDH) product different BD isomers. Catalyzing mechanisms of meso- and S,S-BDH have been understood with the assistance of their X-ray crystal structures. However, the lack of structural data for R,R-BDH restricts the integral understanding of the catalytic mechanism of BDHs. In this study, we successfully crystallized and solved the X-ray crystal structure of Bacillus subtilis R,R-BDH. A zinc ion was found locating in the catalytic center and coordinated by Cys37, His70 and Glu152, helping to stabilize the chiral substrates observed in the predicted molecular docking model. The interaction patterns of different chiral substrates in the molecular docking model explained the react priority measured by the enzyme activity assay of R,R-BDH. Site-directed mutation experiments determined that the amino acids Cys37, Thr244, Ile268 and Lys340 are important in the catalytically active center. The structural information of R,R-BDH presented in this study accomplished the understanding of BDHs catalytic mechanism and more importantly provides useful guidance for the directional engineering of R,R-BDH to obtain high-purity monochiral BD and AC.     

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

Dihydrolipoamide dehydrogenase (DHLDH), dihydrolipoamide acetyltransferase (DHLTA), and acetoin: 2,6-dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) were purified from acetoin-grown cells of Pelobacter carbinolicus. DHLDH had a native Mr of 110,000, consisted of two identical subunits of Mr 54,000, and reacted only with NAD(H) as a coenzyme. The N-terminal amino acid sequence included the flavin adenine dinucleotide-binding site and exhibited a high degree of homology to other DHLDHs. DHLTA had a native Mr of greater than 500,000 and consisted of subunits identical in size (Mr 60,000). The enzyme was highly sensitive to proteolytic attack. During limited tryptic digestion, two major fragments of Mr 32,500 and 25,500 were formed. Ao:DCPIP OR consisted of two different subunits of Mr 37,500 and 38,500 and had a native Mr in the range of 143,000 to 177,000. In vitro in the presence of DCPIP, it catalyzed a thiamine pyrophosphate-dependent oxidative-hydrolytic cleavage of acetoin, methylacetoin, and diacetyl. The combination of purified Ao:DCPIP OR, DHLTA, and DHLDH in the presence of thiamine pyrophosphate and the substrate acetoin or methylacetoin resulted in a coenzyme A-dependent reduction of NAD. In the strictly anaerobic acetoin-utilizing bacteria P. carbinolicus, Pelobacter venetianus, Pelobacter acetylenicus, Pelobacter propionicus, Acetobacterium carbinolicum, and Clostridium magnum, the enzymes Ao:DCPIP OR, DHLTA, and DHLDH were induced during growth on acetoin, whereas they were absent or scarcely present in cells grown on a nonacetoinogenic substrate.     

Genetic characterization of the inducible SOS-like system of Bacillus subtilis.

The SOS-like system of Bacillus subtilis consists of several coordinately induced phenomena (e.g., cellular filamentation, prophage induction, and Weigle reactivation of UV-damaged bacteriophage) which are expressed after cellular insult such as DNA damage or inhibition of DNA replication. Mutagenesis of the bacterial chromosome and the development or maintenance of competence also appear to be involved in the SOS-like response in this bacterium. The genetic characterization of the SOS-like system has involved an analysis of (i) the effects of various DNA repair mutations on the expression of inducible phenomena and (ii) the tsi-23 mutation, which renders host strains thermally inducible for each of the SOS-like functions. Bacterial filamentation was unaffected by any of the DNA repair mutations studied. In contrast, the induction of prophage after thermal or UV pretreatment was abolished in strains carrying the recE4, recA1, recB2, or recG13 mutation. The Weigle reactivation of UV-damaged bacteriophage was also inhibited by the recE4, recA1, recB2, or recG13 mutation, whereas levels of Weigle reactivation were lower in strains which carried the uvrA42, polA5, or rec-961 mutation than in the DNA repair-proficient strain. Strains which carried the recE4 mutation were incapable of chromosomal DNA-mediated transformation, and the frequency of this event was decreased in strains carrying the recA1, recB2, or tsi-23 mutation. Plasmid DNA transformation efficiency was decreased only in strains carrying the tsi-23 mutation in addition to the recE4, recA1, or recB2 mutation. The results indicate that the SOS-like system of B. subtilis is regulated at different levels by two or more gene products. In this report, the current data regarding the genetic regulation of inducible phenomena are summarized, and a model is proposed to explain the mechanism of SOS-like induction in B. subtilis.     

Genetic and biochemical characterization of kirromycin resistance mutations in Bacillus subtilis.

Spontaneous mutations causing resistance to the EF-Tu-specific antibiotic kirromycin have been isolated and mapped in Bacillus subtilis. Three-factor transductional and transformational crosses have placed the kir locus proximal to ery-1 and distal to strA (rpsL) and several mutations affecting elongation factors EF-G and EF-Tu, in the order: cysA strA [fus-1/ts-6(EF-G)] [ts-5(EF-Tu)] kir ery-1 spcA. Purified EF-Tu from mutant strains is more resistant to kirromycin as measured by in vitro protein synthesis and also shows a more acidic isoelectric point than wild-type EF-Tu. This indicates that the kir locus is the genetic determinant (tuf) for EF-Tu and that there is a single active gene for this enzyme in B. subtilis.     

Cloning and characterization of the Alcaligenes eutrophus 2-oxoglutarate dehydrogenase complex

Abstract Nucleotide sequence analysis of a 3.3-kb genomic Eco RI fragment and of relevant subfragments of a genomic 13.2-kb Sma I fragment of Alcaligenes eutrophus , which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative −35 / −10 promoter in the order odhA, odhB , and odhL . In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous expression of the A. eutrophus odh genes in E. coli XL1-Blue and in the kgdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.     

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.     

Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli. I: Purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium

Escherichia coli containing the Bacillus subtilis glucose dehydrogenase gene on a plasmid (prL7) was used to produce the enzyme in high quantities. Gluc-DH-S was purified from the cell extract by (NH4)2SO4-precipitation, ion-exchange chromatography and Triazine-dye chromatography to a specific activity of 375 U/mg. The enzyme was apparently homogenous on SDS-PAGE with a subunit molecular mass of 31.5 kDa. Investigation of Gluc-DH-S was performed for comparison with the corresponding properties of Gluc-DH-M. The limiting Michaelis constant at pH 8.0 for NAD+ is Ka = 0.11 mM and for D-glucose Kb = 8.7 mM. The dissociation constant for NAD+ is Kia = 17.1 mM. Similar to Gluc-DH-M, Gluc-DH-S is inactivated by dissociation under weak alkaline conditions at pH 9.0. Complete reactivation is attained by readjustment to pH 6.5. Ultraviolet absorption, fluorescence and CD-spectra of native Gluc-DH-S, as well as fluorescence- and CD-backbone-spectra of the dissociated enzyme were nearly identical to the corresponding spectra of Gluc-DH-M. The aromatic CD-spectrum of dissociated Gluc-DH-S was different, representing a residual ellipticity of tryptophyl moieties in the 290-310 nm region. Density gradient centrifugation proved that this behaviour is due to the formation of inactive dimers in equilibrium with monomers after dissociation. In comparison to Gluc-DH-M, the kinetics of inactivation as well as the time-dependent change of fluorescence intensity at pH 9.0 of Gluc-DH-S showed a higher velocity and a changed course of the dissociation process.     

A Bacillus subtilis malate dehydrogenase gene.

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation.