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The Phosphoketolase from Bifidobacterium Lactis Biochemistry, Genetics, and Phylogeny

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The Phosphoketolase from Bifidobacterium Lactis Biochemistry, Genetics, and Phylogeny Research Collection Doctoral Thesis The phosphoketolase from Bifidobacterium lactis Biochemistry, genetics, and phylogeny Author(s): Rohr, Lukas Martin Publication Date: 2003 Permanent Link: https://doi.org/10.3929/ethz-a-004630490 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETHno. 15285 The Phosphoketolase from Bifidobacterium lactis: Biochemistry, Genetics, and Phylogeny A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH (ETHZ) for the degree of Doctor of Sciences presented by Lukas Martin Rohr dipl. Lm.-Ing. ETH born October 16, 1973 citizen of Hunzenschwil (AG) accepted on the recommendation of Prof. Dr. Michael Teuber, examiner PD Dr. Leo Meile, co-examiner Dr. Fabrizio Arigoni, co-examiner Zurich, 2003 Contents Abbreviations Summary Zusammenfassung 1 Introduction 1 1.1 The genus Bifidobacterium at a glance 1 1.2 Bifidobacteria and health promotion 2 1.3 The hexose metabolism of bifidobacteria 3 1.4 Phosphoketolases and thiamin diphosphate (ThDP)-dependent enzymes 5 1.5 Phosphoketolases in microorganisms 7 1.6 Aim of this study 15 2 Materials and Methods 17 2.1 Microbial strains and growth conditions 17 2.2 Plasmids and oligonucleotides 18 2.3 Protein techniques 21 2.3.1 Preparation of cell-free extracts 21 2.3.2 Protein determination 21 2.3.3 Anion exchange and gel filtration chromatography 21 2.3.4 Phosphoketolase assays 22 2.3.5 Polyacrylamide gel electrophoresis 23 2.3.6 N-terminal amino acid sequence analysis 23 2.3.7 Expression induction of recombinant phosphoketolase genes and localisation of the target proteins in Escherichia coli 24 2.4 DNA techniques 24 2.4.1 Methods of DNA isolation 24 2.4.2 Quantification of DNA 26 2.4.3 Agarose gel electrophoresis 26 2.4.4 Restriction endonuclease digestions 26 2.4.5 Polymerase chain reaction (PCR) 27 2.4.6 Ligation of DNA fragments 27 2.4.7 Transformation procedures 28 2.4.8 Hybridisation techniques 28 2.4.9 Nucleotide sequence determination and analysis 30 2.5 RNA techniques 31 2.5.1 Isolation of total RNA from Bifidobacterium lactis 31 2.5.2 Quantification of RNA 31 2.5.3 Northern and slot blot analysis 32 2.5.4 Primer extension analysis 32 I 3 Results 33 3.1 Purification of a protein with fructose 6-phosphate phosphoketolase activity from Bifidobacterium lactis 33 3.2 Identification and cloning of the phosphoketolase gene {xfp) from Bifidobacterium lactis 35 3.3 DNA sequence of the Bifidobacterium lactis xfp gene and its adj acent region 37 3.4 Heterologous expression of the Bifidobacterium lactis phosphoketolase gene in Escherichia coli 41 3.5 Enzymatic characterisation of the native and recombinant Bifidobacterium lactis phosphoketolase 45 3.5.1 Dual substrate specificity 45 3.5.2 Heat stability 46 3.5.3 Thiamin diphosphate as a stabiliser of recombinant phosphoketolase 48 3.6 Expression analysis of'theBifidobacterium lactisxfp gene 50 3.6.1 Northern and primer extension analysis 50 3.6.2 Is there a regulation of xfp transcription and/or translation m Bifidobacterium lactis? 52 3.7 Substitution of xylulose 5-phosphate used as a substrate in phosphoketolase assays 54 3.8 The heterogeneity of bifidobacterial phosphoketolases 55 3.9 From hexameric to dimeric phosphoketolase by mechanical treatment? 57 3.10 Prevalence of the xfp gene in microbial genomes 59 3.11 Signature patterns within the Xfp phosphoketolase family 66 3.12 Attempts of cloning the Bifidobacterium lactis xfp gene in Propionibacterium freudenreichii subsp. freudenreichii 68 4 Discussion 73 4.1 The Bifidobacterium lactis phosphoketolase is a dual substrate enzyme 73 4.2 The Bifidobacterium lactis phosphoketolase gene and its expression in Escherichia coli 74 4.3 The xfp gene is not transcriptionally regulated in Bifidobacterium lactis 76 4.4 The bifidobacterial phosphoketolases: a manifold uniformity? 78 4.5 Phylogenetic distribution of xfp family genes in the microbial world 81 4.6 Horizontal gene transfer or evolutionary gene loss? 89 4.7 Conclusion and outlook 93 5 References 95 Appendix 107 Acknowledgements Curriculum vitae II Abbreviations aa amino acid(s) ADP adenosine diphosphate ATP adenosine triphosphate DTT 1,4-dithio-DL-threitol EDTA ethylenediaminetetraacetic acid disodium salt F6P D-fructose 6-phosphate F6PPK fructose 6-phosphate phosphoketolase IPTG isopropylyö-D-thiogalactopyranoside NAD(P)+ nicotinamide adenine dinucleotide (phosphate), oxidised form NAD(P)H nicotinamide adenine dinucleotide (phosphate), reduced form nt nucleotide(s) OD optical density ORF open reading frame PCR polymerase chain reaction PMSF phenylmethylsulfonyl fluoride RT room temperature SDS sodium dodecyl sulphate ThDP thiamin diphosphate Tris tri s(hydroxymethyl)aminomethane X5P D-xylulose 5-phosphate X5PPK xylulose 5-phosphate phosphoketolase Xfp D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase III Summary Bifidobacteria belong to the predominant microflora of the human and animal intestine. The attribution of various health-promoting qualities to them has markedly increased the scientific and economic interest in these Gram-positive anaerobes. Besides their application as probiotic food supplements, the ability of bifidobacteria to degrade a wide range of complex carbo¬ hydrates predestines them as target organisms for prebiotic substances. The fermentation of hexoses by bifidobacteria follows a characteristic pathway known as the fructose 6-phosphate (F6P) shunt with its characteristic key enzyme fructose 6-phosphate phosphoketolase (F6PPK), which converts F6P to acetyl phosphate and erythrose 4-phosphate. Phosphoketo¬ lases splitting xylulose 5-phosphate (X5P) play a major role in heterolactic fermentation, but enzymes of both specificities have also been described in other microorganisms. Since mole¬ cular data on any phosphoketolase were lacking at the beginning of this study, the main goal was the biochemical and genetic investigation of this thiamin diphosphate (ThDP)-dependent enzyme. A protein exhibiting F6PPK activity was purified from Bifidobacterium lactis to a specific activity of 4.28 U per mg of protein. Its molecular mass was estimated to 550 kDa, while the subunit size upon sodium dodecyl sulphate-polyacrylamide gel electrophoresis was approxi¬ mately 90 kDa, suggesting a homohexameric quarternary structure for the native protein. The enzyme was revealed to be a dual substrate D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase (Xfp) with apparent Km values of 45 and 10 mM for X5P and F6P, respec¬ tively. The xfp gene was identified on the B. lactis chromosome using degenerated oligonuc¬ leotide probes deduced from the N-terminal amino acid sequence of both the native and de¬ natured enzyme. A two-step procedure resulted in the cloning of a 4,123-bp segment of chromosomal DNA containing an open reading frame (ORF) coding for a 825-amino acid protein, of which the N terminus and the calculated size (92,529 Da) corresponded to the pro¬ perties of the purified protein. On this cloned fragment, the xfp gene is flanked by two trun¬ cated ORFs assigned to phosphotransacetylase (Pta) and guanosine monophosphate synthe¬ tase (GuaA), respectively. Whilst all attempts to clone the B. lactis xfp gene in Propionibacterium freudenreichii subsp. freudenreichii failed, final evidence of the enzyme to possess dual substrate phosphoketolase activity was obtained by the heterologous expres¬ sion in Escherichia coli. The recombinant Xfp protein was found to differ from the native en¬ zyme in respects of its heat sensitivity; moreover, the addition of the cofactor ThDP was cru¬ cial for considerable activity and medium-term storage of the recombinant enzyme. V As shown by Northern blot experiments, the xfp gene is transcribed in B. lactis as a mono- cistronic operon. The transcription start point was located 96 nucleotides upstream of the translation initiation codon. The F6PPK activity in crude extracts was not altered by growth of B. lactis cells on four different sugars, whereas it was halfed upon transition from the ex¬ ponential to the stationary growth phase. However, no change of the xfp transcript concentra¬ tion could be detected simultaneously, making a regulation on that level improbable. Gel filtration chromatography experiments with the crude extracts of twelve Bifidobacterium species and of Gardnerella vaginalis revealed a second phosphoketolase activity of approxi¬ mately 160 kDa besides the 550-kDa enzyme in most samples. Further analyses supported the assumption that both proteins consist of the same monomer encoded by the xfp gene, whereby the hexamer is prone to decay to a dimeric protein of 160 kDa under certain conditions. Database searches led to the identification of 63 hypothetical proteins with high similarity to the B. lactis Xfp amino acid sequence in the genomes of 55 different microorganisms of a broad taxonomic range, including e.g. representatives from the proteobacteria, cyanobacteria, and yeasts, but not from archaeal species. All of these sequences share a protein motif com¬ mon for ThDP enzymes and a transketolase signature; furthermore, two new Xfp phosphoke¬ tolase consensus patterns are proposed. From the results of comparative sequence analyses, combined with the available biochemical data, it can be speculated that the Xfp protein is a microbial ,ur-protein' which is still essential within the carbohydrate catabolism of some ta¬ xons, whereas in
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