Altenbuchner J

References (16)

Title : A novel esterase subfamily with alpha\/beta-hydrolase fold suggested by structures of two bacterial enzymes homologous to l-homoserine O-acetyl transferases - Tolzer_2016_FEBS.Lett_590_174
Author(s) : Tolzer C , Pal S , Watzlawick H , Altenbuchner J , Niefind K
Ref : FEBS Letters , 590 :174 , 2016
Abstract : MekB from Pseudomonas veronii and CgHle from Corynebacteriumglutamicum belong to the superfamily of alpha/beta-hydrolase fold proteins. Based on sequence comparisons, they are annotated as homoserine transacetylases in popular databases like UNIPROT, PFAM or ESTHER. However, experimentally, MekB and CgHle were shown to be esterases that hydrolyse preferentially acetic acid esters. We describe the x-ray structures of these enzymes solved to high resolution. The overall structures confirm the close relatedness to experimentally validated homoserine acetyl transferases, but simultaneously the structures exclude the ability of MekB and CgHle to bind homoserine and acetyl-CoA. Insofar the MekB and CgHle structures suggest dividing the homoserine transacetylase family into subfamilies, namely genuine acetyl transferases and acetyl esterases with MekB and CgHle as constituting members of the latter.
ESTHER : Tolzer_2016_FEBS.Lett_590_174
PubMedSearch : Tolzer_2016_FEBS.Lett_590_174
PubMedID: 26787467
Gene_locus related to this paper: 9psed-q0mrg5

Title : Crystallization and preliminary crystallographic analysis of cgHle, a homoserine acetyltransferase homologue, from Corynebacterium glutamicum - Tolzer_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_34
Author(s) : Tolzer C , Pal S , Watzlawick H , Altenbuchner J , Niefind K
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 65 :34 , 2009
Abstract : CgHle is an enzyme that is encoded by gene cg0961 from Corynebacterium glutamicum. The physiological function of cgHle is so far unclear. Bioinformatic annotations based on sequence homology indicated that cgHle may be an acetyl-CoA:homoserine acetyl transferase and as such may be involved in methionine biosynthesis, but recent evidence has shown that it is an esterase that catalyzes the hydrolysis of acetyl esters. Here, the crystallization of cgHle in two orthorhombic crystal forms, a trigonal crystal form and a monoclinic crystal form is described. The trigonal crystals have a solvent content of 83.7%, which is one of the highest solvent contents ever found for protein crystals. One of the orthorhombic crystals diffracted X-rays to at least 1.2 A resolution.
ESTHER : Tolzer_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_34
PubMedSearch : Tolzer_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_34
PubMedID: 19153452
Gene_locus related to this paper: corgl-CGL0839

Title : Design of a secondary alcohol degradation pathway from Pseudomonas fluorescens DSM 50106 in an engineered Escherichia coli - Kirschner_2007_Appl.Microbiol.Biotechnol_75_1095
Author(s) : Kirschner A , Altenbuchner J , Bornscheuer UT
Ref : Applied Microbiology & Biotechnology , 75 :1095 , 2007
Abstract : The genes encoding an alcohol dehydrogenase, Baeyer-Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (C(n)) to the corresponding primary alcohols (C(n-2)) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30 degrees C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons.
ESTHER : Kirschner_2007_Appl.Microbiol.Biotechnol_75_1095
PubMedSearch : Kirschner_2007_Appl.Microbiol.Biotechnol_75_1095
PubMedID: 17347816

Title : Degradation of alkyl methyl ketones by Pseudomonas veronii MEK700 - Onaca_2007_J.Bacteriol_189_3759
Author(s) : Onaca C , Kieninger M , Engesser KH , Altenbuchner J
Ref : Journal of Bacteriology , 189 :3759 , 2007
Abstract : Pseudomonas veronii MEK700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. The strain is able to grow on 2-butanone and 2-hexanol. The genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-Tn5495, and cloned in Escherichia coli. DNA sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. The deduced amino acid sequence of the first gene, mekA, had high similarity to Baeyer-Villiger monooxygenases; the protein of the second gene, mekB, had similarity to homoserine acetyltransferases; the third gene, mekR, encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, and the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the mini-transposon abolished growth of P. veronii MEK700 on 2-butanone and 2-hexanol. The involvement of mekR in methyl ketone degradation was observed by heterologous expression of mekA and mekB in Pseudomonas putida. A fragment containing mekA and mekB on a plasmid was not sufficient to allow P. putida KT2440 to grow on 2-butanone. Not until all three genes were assembled in the recombinant P. putida was it able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase activity of MekA was clearly demonstrated by incubating a mekB transposon insertion mutant of P. veronii with 2-butanone. Hereby, ethyl acetate was accumulated. To our knowledge, this is the first time that ethyl acetate by gas chromatographic analysis has been definitely demonstrated to be an intermediate of MEK degradation. The mekB-encoded protein was heterologously expressed in E. coli and purified by immobilized metal affinity chromatography. The protein exhibited high esterase activity towards short chain esters like ethyl acetate and 4-nitrophenyl acetate.
ESTHER : Onaca_2007_J.Bacteriol_189_3759
PubMedSearch : Onaca_2007_J.Bacteriol_189_3759
PubMedID: 17351032
Gene_locus related to this paper: 9psed-q0mrg5

Title : Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106 - Hildebrandt_2002_Appl.Microbiol.Biotechnol_59_483
Author(s) : Hildebrandt P , Musidlowska A , Bornscheuer UT , Altenbuchner J
Ref : Applied Microbiology & Biotechnology , 59 :483 , 2002
Abstract : Sequencing of a genomic library prepared from Pseudomonas fluorescens DSM 50106 identified an orf showing 29% identity to a C alpha-dehydrogenase of Pseudomonas paucimobilis and high homology to several sequences with unknown functions derived from genome projects. The corresponding gene adhF1 encodes a dehydrogenase of 296 amino acids with a calculated molecular mass of 31.997 kDa. The gene was functionally expressed in E. coli using a rhamnose inducible expression system. The resulting recombinant enzyme was active in the pH range 6-10 (best pH 8) and at 5-25 degrees C. This dehydrogenase converts cyclic ketones to the corresponding alcohols utilizing the cofactor NADH. The highest activity was found for cyclohexanone. The enzyme also exhibits high stereoselectivity in the desymmetrization of the prochiral ketone acetophenone, producing optically pure ( R)-alpha-phenyl ethanol (>99%ee) at high conversion (95%).
ESTHER : Hildebrandt_2002_Appl.Microbiol.Biotechnol_59_483
PubMedSearch : Hildebrandt_2002_Appl.Microbiol.Biotechnol_59_483
PubMedID: 12172614

Title : Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones - Khalameyzer_1999_Appl.Environ.Microbiol_65_477
Author(s) : Khalameyzer V , Fischer I , Bornscheuer UT , Altenbuchner J
Ref : Applied Environmental Microbiology , 65 :477 , 1999
Abstract : A genomic library of Pseudomonas fluorescens DSM 50106 in a lambdaRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using alpha-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43 degreesC. The showed highest purified enzyme activities towards lactones. The activity increased from gamma-butyrolactone (18.1 U/mg) to epsilon-caprolactone (21.8 U/mg) to delta-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.
ESTHER : Khalameyzer_1999_Appl.Environ.Microbiol_65_477
PubMedSearch : Khalameyzer_1999_Appl.Environ.Microbiol_65_477
PubMedID: 9925571
Gene_locus related to this paper: psefl-estF1

Title : Directed evolution of an esterase: screening of enzyme libraries based on pH-indicators and a growth assay - Bornscheuer_1999_Bioorg.Med.Chem_7_2169
Author(s) : Bornscheuer UT , Altenbuchner J , Meyer HH
Ref : Bioorganic & Medicinal Chemistry , 7 :2169 , 1999
Abstract : In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed. Mutations were introduced using the mutator strain Epicurian coli XL1-Red. Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid. A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media. By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5).
ESTHER : Bornscheuer_1999_Bioorg.Med.Chem_7_2169
PubMedSearch : Bornscheuer_1999_Bioorg.Med.Chem_7_2169
PubMedID: 10579522

Title : Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones - Bornscheuer_1998_Biotechnol.Bioeng_58_554
Author(s) : Bornscheuer UT , Altenbuchner J , Meyer HH
Ref : Biotechnol Bioeng , 58 :554 , 1998
Abstract : The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.
ESTHER : Bornscheuer_1998_Biotechnol.Bioeng_58_554
PubMedSearch : Bornscheuer_1998_Biotechnol.Bioeng_58_554
PubMedID: 10099292

Title : Structural investigation of the cofactor-free chloroperoxidases - Hofmann_1998_J.Mol.Biol_279_889
Author(s) : Hofmann B , Tolzer S , Pelletier I , Altenbuchner J , van Pee KH , Hecht HJ
Ref : Journal of Molecular Biology , 279 :889 , 1998
Abstract : The structures of cofactor-free haloperoxidases from Streptomyces aureofaciens, Streptomyces lividans, and Pseudomonas fluorescens have been determined at resolutions between 1.9 A and 1.5 A. The structures of two enzymes complexed with benzoate or propionate identify the binding site for the organic acids which are required for the haloperoxidase activity. Based on these complexes and on the structure of an inactive variant, a reaction mechanism is proposed for the halogenation reaction with peroxoacid and hypohalous acid as reaction intermediates. Comparison of the structures suggests that a specific halide binding site is absent in the enzymes but that hydrophobic organic compounds may fit into the active site pocket for halogenation at preferential sites.
ESTHER : Hofmann_1998_J.Mol.Biol_279_889
PubMedSearch : Hofmann_1998_J.Mol.Biol_279_889
PubMedID: 9642069
Gene_locus related to this paper: psefl-cpoF , strau-brpa2 , strau-cpoT , strli-cpoL

Title : Analysis of a new dimeric extradiol dioxygenase from a naphthalenesulfonate-degrading sphingomonad - Heiss_1997_Microbiology_143_1691
Author(s) : Heiss G , Muller C , Altenbuchner J , Stolz A
Ref : Microbiology , 143 ( Pt 5) :1691 , 1997
Abstract : A new extradiol dioxygenase was cloned by screening a gene bank from the naphthalenesulfonate-degrading bacterial strain BN6 for colonies with 2,3-dihydroxybiphenyl dioxygenase (DHBPDO) activity. A 1.6 kb DNA fragment was sequenced and an ORF of 954 bp identified. Comparison of the deduced amino acid sequence of DHBPDO II from strain BN6 with previously published sequences showed the closest relationship to a metapyrocatechase (MpcII) from Alcaligenes eutrophus JMP 222. Thus, the enzyme was only distantly related to the main groups of catechol 2,3-dioxygenases or DHBPDOs. The dioxygenase was expressed using a T7 expression vector and the enzymic characteristics of the protein were examined. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-methylcatechol, 4-fluorocatechol and 1,2-dihydroxynaphthalene. Comparison of the UV/visible spectrum of the product formed from 3,5-dichlorocatechol with previous reports suggested that this substrate is oxidized by different extradiol dioxygenases either by proximal or distal ring cleavage. The enzyme required Fe2+ for maximal activity. In contrast to most other extradiol dioxygenases, the enzyme consisted of only two identical subunits.
ESTHER : Heiss_1997_Microbiology_143_1691
PubMedSearch : Heiss_1997_Microbiology_143_1691
PubMedID: 9168618
Gene_locus related to this paper: 9sphn-q1nha7

Title : Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360 - Schmid_1997_J.Bacteriol_179_53
Author(s) : Schmid A , Rothe B , Altenbuchner J , Ludwig W , Engesser KH
Ref : Journal of Bacteriology , 179 :53 , 1997
Abstract : The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K.-H. Engesser, V. Strubel, K. Christoglou, P. Fischer, and H. G. Rast, FEMS Microbiol. Lett. 65:205-210, 1989; V. Strubel, Ph.D. thesis, University of Stuttgart, Stuttgart, Germany, 1991; V. Strubel, H. G. Rast, W. Fietz, H.-J. Knackmuss, and K.-H. Engesser, FEMS Microbiol. Lett. 58:233-238, 1989). Two 2,3-dihydroxybiphenyl-1,2-dioxygenases (BphC1 and BphC2) and one catechol-2,3-dioxygenase (C23O) were shown to be expressed in Terrabacter sp. strain DPO360 growing with dibenzofuran as a sole source of carbon and energy. These enzymes exhibited strong sensitivity to oxygen. They were purified to apparent homogeneity as homodimers (BphC and BphC2) and as a homotetrameric catechol-2,3-dioxygenase (C23O). According to their specificity constants kcat/Km, both BphC1 and BphC2 were shown to be responsible for the cleavage of 2,2',3-trihydroxybiphenyl, the first metabolite in dibenzofuran mineralization along the angular dioxygenation pathway. With this substrate, BphC2 exhibited a considerably higher kcat/Km, value (183 microM/min) than BphC1 (29 microM/min). Catechol-2,3-dioxygenase was recognized to be not involved in the ring cleavage of 2,2',3-trihydroxybiphenyl (kcat/Km, 1 microM/min). Analysis of deduced amino acid sequence data of bphC1 revealed 36% sequence identity to nahC from Pseudomonas putida PpG7 (S. Harayama and M. Rekik, J. Biol. Chem. 264:15328-15333, 1989) and about 40% sequence identity to various bphC genes from different Pseudomonas and Rhodococcus strains. In addition, another 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (bphC3) was cloned from the genome of Terrabacter sp. strain DPO360. Expression of this gene, however, could not be detected in Terrabacter sp. strain DPO360 after growth with dibenzofuran.
ESTHER : Schmid_1997_J.Bacteriol_179_53
PubMedSearch : Schmid_1997_J.Bacteriol_179_53
PubMedID: 8981980

Title : A bacterial esterase is homologous with non-haem haloperoxidases and displays brominating activity - Pelletier_1995_Microbiology_141_459
Author(s) : Pelletier I , Altenbuchner J
Ref : Microbiology , 141 :459 , 1995
Abstract : Screening GenBank indicated that an esterase from Pseudomonas fluorescens had high sequence similarity with bacterial non-haem haloperoxides. However, this homology was limited to two distinct domains of the published esterase sequence. As errors in the published sequence were suspected, the esterase gene was sequenced again. The revised sequence displayed between 40 and 50% identical amino acids with the haloperoxidases, but distributed along the whole sequence. In addition to the structural homologies with haloperoxidases, the esterase also displayed functional homology. The recombinant esterase, purified from Escherichia coli cells, was capable of both ester hydrolysis and halogenation, as detected in situ by the formation of bromophenol blue or spectrophotometrically by the bromination of monochlorodimedon. The esterase is thus a bifunctional enzyme. The sequence analysis and the biochemical investigations show that the esterase belongs to the haloperoxidase family. It also possessed, however, a typical feature of serine-hydrolases, namely the consensus motif Gly-X-Ser-X-Gly around the active serine of the catalytic triad. By alignment of the esterase with different serine-hydrolase sequences, it was possible to identify the other two residues of the triad. The triad comprised the residues Ser95, Asp223 and His252. Interestingly, a structurally equivalent catalytic triad was also identified in the sequences of all bacterial non-haem haloperoxidases, in highly conserved domains. The presence of a catalytic triad in haloperoxidases is expected to be important in the mechanism of halogenation.
ESTHER : Pelletier_1995_Microbiology_141_459
PubMedSearch : Pelletier_1995_Microbiology_141_459
PubMedID: 7704276
Gene_locus related to this paper: psefl-este

Title : A catalytic triad is required by the non-heme haloperoxidases to perform halogenation - Pelletier_1995_Biochim.Biophys.Acta_1250_149
Author(s) : Pelletier I , Altenbuchner J , Mattes R
Ref : Biochimica & Biophysica Acta , 1250 :149 , 1995
Abstract : The bacterial non-heme haloperoxidases are highly related to an esterase from Pseudomonas fluorescens, at structural and functional levels. Both types of enzymes displayed brominating activity and esterase activity. The presence of the serine-hydrolase motif Gly-X-Ser-X-Gly, in the esterase as well as in all aligned haloperoxidase sequences, strongly suggested that they belong to the serine-hydrolase family. Sequence alignment with several serine-hydrolases and secondary structure superimposition revealed the striking conservation of structural features characterising the alpha/beta-hydrolase fold structure in all haloperoxidases. These structural predictions allowed us to identify a potential catalytic triad in haloperoxidases, perfectly matching the triad of all aligned serine-hydrolases. The structurally equivalent triad in the chloroperoxidase CPO-P comprised the amino acids Serine 97, Aspartic acid 229 and Histidine 258. The involvement of this catalytic triad in halogenation was further assessed by inhibition studies and site-directed mutagenesis. Inactivation of CPO-P by PMSF and DEPC strongly suggested that the serine residue from the serine-hydrolase motif and an histidine residue are essential for halogenation, similar to that demonstrated for typical serine-hydrolases. By site-directed mutagenesis of CPO-P, Ser-97 was exchanged against alanine or cysteine, Asp-229 against alanine and His-258 against glutamine. Western blot analysis indicated that each mutant gene was efficiently expressed. Whereas the mutant S97C conserved a very low residual activity, each other mutant S97A, D229A or H258Q was totally inactive. This study gives the direct demonstration of the requirement of a catalytic triad in the halogenation mechanism.
ESTHER : Pelletier_1995_Biochim.Biophys.Acta_1250_149
PubMedSearch : Pelletier_1995_Biochim.Biophys.Acta_1250_149
PubMedID: 7632719

Title : Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene - Bantleon_1994_J.Bacteriol_176_2339
Author(s) : Bantleon R , Altenbuchner J , van Pee KH
Ref : Journal of Bacteriology , 176 :2339 , 1994
Abstract : For the first time, a halogenating enzyme which is not known to produce halogenated metabolites has been isolated from a bacterial strain. The gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyces lividans TK64 was cloned, and its gene product was characterized. S. lividans TK64 produced only very small amounts of the enzyme. After cloning of the gene into Streptomyces aureofaciens T24-88, the enzyme was overexpressed up to 3,000-fold. Based on the overexpression, a simple purification procedure using acid precipitation and hydrophobic interaction chromatography was developed. Thus, 54 mg of homogeneous CPO-L could be obtained from 27 g (wet weight) of mycelium. The native enzyme has a molecular weight of 64,000 and consists of two identical subunits. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts. CPO-L showed cross-reaction with antibodies raised against the nonheme chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S. aureofaciens T24. CPO-L exhibits substrate specificity only for chlorination, not for bromination. Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is brominated and chlorinated. The functional chloroperoxidase gene was located on a 1.9-kb SalI DNA fragment. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide of 276 amino acids. The overall identity of the amino acid sequence to that of chloroperoxidase from P. pyrrocinia was 71%, whereas that to bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 was only 42%
ESTHER : Bantleon_1994_J.Bacteriol_176_2339
PubMedSearch : Bantleon_1994_J.Bacteriol_176_2339
PubMedID: 8157602
Gene_locus related to this paper: strli-cpoL

Title : Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification - Pelletier_1994_Microbiology_140_509
Author(s) : Pelletier I , Pfeifer O , Altenbuchner J , van Pee KH
Ref : Microbiology , 140 :509 , 1994
Abstract : The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64. The cloned bromoperoxidase was over-produced up to 2800-fold by the S. lividans TK64 transformant. By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed. Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the bpoA1 gene on a 2.1 kb BamHI-HindIII fragment. The nucleotide sequence of the 2.1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1. Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.
ESTHER : Pelletier_1994_Microbiology_140_509
PubMedSearch : Pelletier_1994_Microbiology_140_509
PubMedID: 8012573
Gene_locus related to this paper: strau-brpa1

Title : Molecular cloning and sequencing of a non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762 - Pfeifer_1992_J.Gen.Microbiol_138_1123
Author(s) : Pfeifer O , Pelletier I , Altenbuchner J , van Pee KH
Ref : J Gen Microbiol , 138 :1123 , 1992
Abstract : A bromoperoxidase gene (bpoT), recently cloned from Streptomyces aureofaciens Tu24, was used as a probe in Southern blot hybridization of total DNA from S. aureofaciens ATCC 10762. A single SstI fragment of 5.4 kb was detected, which was cloned via an enriched gene library into Escherichia coli. The functional bromoperoxidase gene was located on a 2.1 kb BamHI-HindIII fragment by subcloning into S. lividans TK64, using the multicopy plasmid pIJ486. The enzyme was overproduced in S. lividans TK64 (up to 30,000 times compared to S. aureofaciens ATCC 10762) and showed the same electrophoretic and immunological properties as the bromoperoxidase BPO-A2 purified from S. aureofaciens ATCC 10762. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide with the same M(r) and N-terminal amino acid sequence as the purified subunit of BPO-A2.
ESTHER : Pfeifer_1992_J.Gen.Microbiol_138_1123
PubMedSearch : Pfeifer_1992_J.Gen.Microbiol_138_1123
PubMedID: 1527491
Gene_locus related to this paper: strau-brpa2