BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B 12 by Propionibacterium freudenreichii

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dc.contributor.author Deptula, Paulina
dc.contributor.author Kylli, Petri Tapio
dc.contributor.author Chamlagain, Bhawani Shankar
dc.contributor.author Holm, Liisa Ulrika Teodora
dc.contributor.author Kostiainen, Risto Kalervo
dc.contributor.author Piironen, Vieno Irene
dc.contributor.author Savijoki, Kirsi Kristiina
dc.contributor.author Varmanen, Pekka Kristian
dc.date.accessioned 2017-05-04T12:00:01Z
dc.date.available 2017-05-04T12:00:01Z
dc.date.issued 2015-11-23
dc.identifier.citation Deptula , P , Kylli , P T , Chamlagain , B S , Holm , L U T , Kostiainen , R K , Piironen , V I , Savijoki , K K & Varmanen , P K 2015 , ' BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B 12 by Propionibacterium freudenreichii ' , Microbial Cell Factories , vol. 14 , 186 . https://doi.org/10.1186/s12934-015-0363-9
dc.identifier.other PURE: 56670724
dc.identifier.other PURE UUID: c7c6d05a-f3c9-440d-94d2-726e88bd5c0b
dc.identifier.other WOS: 000365270200001
dc.identifier.other Scopus: 84947749338
dc.identifier.other ORCID: /0000-0002-4508-3060/work/48395646
dc.identifier.other ORCID: /0000-0003-2454-4574/work/29819412
dc.identifier.other ORCID: /0000-0002-7939-6866/work/28420239
dc.identifier.other ORCID: /0000-0002-8854-3573/work/37697970
dc.identifier.other ORCID: /0000-0003-2831-3503/work/85519186
dc.identifier.uri http://hdl.handle.net/10138/182611
dc.description.abstract Background Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B 12 . Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B 12 by microbial fermentation. Active vitamin B 12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B 12 . Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B 12 production properties. Results Here, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions. Conclusions The herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B 12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B 12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B 12 within food products. Keywords: Propionibacterium freudenreichii ; Cobalamin; B12; DMBI; α-Ribazole; Phosphoribozyltransferase; Nitroreductase; Fusion enzyme; BluB; CobT en
dc.format.extent 12
dc.language.iso eng
dc.relation.ispartof Microbial Cell Factories
dc.rights cc_by
dc.rights.uri info:eu-repo/semantics/openAccess
dc.subject 1182 Biochemistry, cell and molecular biology
dc.title BluB/CobT2 fusion enzyme activity reveals mechanisms responsible for production of active form of vitamin B 12 by Propionibacterium freudenreichii en
dc.type Article
dc.contributor.organization Department of Food and Nutrition
dc.contributor.organization Molecular Dairy Microbiology
dc.contributor.organization Food quality and safety: lipids, vitamins and other bioactive compounds
dc.contributor.organization Faculty of Pharmacy
dc.contributor.organization Division of Pharmaceutical Chemistry and Technology
dc.contributor.organization Biosciences
dc.contributor.organization Computational genomics
dc.contributor.organization Genetics
dc.contributor.organization Institute of Biotechnology
dc.contributor.organization Risto Kostiainen / Principal Investigator
dc.contributor.organization Bioinformatics
dc.contributor.organization Drug Research Program
dc.contributor.organization Food Sciences
dc.description.reviewstatus Peer reviewed
dc.relation.doi https://doi.org/10.1186/s12934-015-0363-9
dc.relation.issn 1475-2859
dc.rights.accesslevel openAccess
dc.type.version publishedVersion
dc.identifier.url http://www.microbialcellfactories.com/content/supplementary/s12934-015-0363-9-s1.pdf

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