Kloosterman, Harmen

English

Harm Kloosterman deed onderzoek naar een familie eiwitten, de Nudix hydrolases, die qua structuur en katalytische activiteit verwant zijn. Het zijn enzymen die de hydrolyse van een fosfaatbinding in een nucleotide kunnen katalyseren. Van twee leden van deze familie onderzocht Kloosterman de specifieke functie. Zo ontdekte hij onder andere dat Nudix hydrolases niet slechts de concentraties van verschillende nucleotiden kunnen reguleren, maar dat ze in bepaalde gevallen ook de functionele eigenschappen van nucleotide bindende eiwitten kunnen reguleren

University of Groningen

   University of GroningenBiochemical and functional characterization of Nudix hydrolase enzymes with novel regulatoryroles in Gram positive methylotrophic bacteriaKloosterman, HarmenIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Kloosterman, H. (2005). Biochemical and functional characterization of Nudix hydrolase enzymes withnovel regulatory roles in Gram positive methylotrophic bacteria. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 25-04-2023           Biochemical and functional characterization of Nudix hydrolase enzymes with novel regulatory roles in Gram positive methylotrophic bacteria                             Harm Kloosterman                                            The study described in this thesis was performed at the Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, The Netherlands.   The research was supported in part by TNO Environmental Research.      RIJKSUNIVERSITEIT GRONINGEN      BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF NUDIX HYDROLASE ENZYMES WITH NOVEL REGULATORY ROLES IN GRAM POSITIVE METHYLOTROPHIC BACTERIA       Proefschrift     ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op vrijdag 28 oktober 2005 om 14.45 uur     door       Harmen Kloosterman  geboren op 6 juli 1964  te Garijp   Promotor:   Prof. Dr. L. Dijkhuizen   Beoordelingscommissie: Prof. Dr. A.J. Driessen      Prof. Dr. J.D. van Elsas      Prof. Dr. J. Kok    5 Contents                                       Aim and outline of thesis  7 Chapter 1 General introduction into Nudix hydrolase proteins   11 Chapter 2 Nicotinoprotein methanol dehydrogenase enzymes in Gram-positive methylotrophic bacteria  25 Chapter 3 Molecular, biochemical and functional characterization of a Nudix hydrolase protein that stimulates activity of a nicotinoprotein alcohol dehydrogenase.  33 Chapter 4 Identification of a magnesium-dependent NAD(P)(H) binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus  53 Chapter 5 A Nudix hydrolase protein with a crucial role in regulation of  Amycolatopsis methanolica plasmid pMEA300 encoded functions   69 Chapter 6 Identification and characterisation of the minimal replicon of the indigenous plasmid pMEA300 of the actinomycete Amycolatopsis  methanolica  85 Chapter 7 (De)Regulation of key enzyme steps in the shikimate pathway and phenylalanine specific pathway of the actinomycete Amycolatopsis methanolica  97 Chapter 8 Summary and concluding remarks  115 Chapter 9 Samenvatting  125  References  133  Dankwoord  149   6     7    Aim and Outline of Thesis   8  Aim and outline of thesis    9At the start of this study no information was available about possible regulatory functions of Nudix (Nucleotide diphosphate linked to some other moiety, X) hydrolase proteins. This protein family was regarded as a group of enzymes necessary to remove potentially toxic or mutagenic compounds from the cell. This is exemplified by the first member of this protein family, MutT of E. coli, which was shown to hydrolyze 8-oxo-dGTP, a potentially mutagenic form of dGTP when used for DNA synthesis. The entire Nudix hydrolase protein family subsequently was referred to as “housecleaning enzymes”. The cloning and characterization of the methanol dehydrogenase (MDH) activator protein (ACT) of Bacillus methanolicus provided the first example of a Nudix hydrolase protein regulating an enzymatic activity involved in primary metabolism. Also a Nudix hydrolase encoding ORF on the indigenous plasmid pMEA300 of Amycolatopsis methanolica was shown to be an important regulator of various plasmid functions (pock formation, autonomous replication and stimulation of transformation frequency). The primary aim of this PhD study was to elucidate the physiological roles, regulatory functions and mechanisms of both Nudix hydrolases, which share significant overall amino acid sequence similarity.  Chapter 1 reviews the current knowledge of the Nudix hydrolase protein family, with emphasis on Nudix hydrolase proteins in microorganisms. Chapter 2 discusses the nicotinoprotein methanol dehydrogenase (MDH) enzymes in Gram-positive bacteria. The MDH enzymes in both B. methanolicus and A. methanolica are nicotinoproteins, with a tightly bound NAD(P)(H) cofactor. The B. methanolicus ACT protein hydrolyzes the NAD(H) cofactor of MDH (chapter 3). A detailed biochemical and mutational analysis of the catalytic function of this NAD(H) cofactor and its binding site in MDH are presented in chapter 4. Chapter 5 reports the identification of the minimal replicon of A. methanolica plasmid pMEA300, revealing that none of the regulatory genes -including the Nudix hydrolase encoding orf192- are essential for replication. The pMEA300 Rep protein shows no similarity with any other Rep protein in databases. The data suggest that pMEA300 belongs to a new family of Rolling Circle Replication plasmids. A functional analysis of the Nudix hydrolase Orf192 protein and other regulatory proteins of pMEA300 is described in chapter 6, revealing their effect on integration, pock formation and transformation frequency. Chapter 7 describes the regulation of key enzymes in the shikimate pathway of A. methanolica. Mutant enzymes affected in feedback inhibition/activation control were isolated by screening for mutant strains resistant to toxic phenylalanine analogs and subsequently characterized. Those mutants could only be isolated in the A. methanolica wild type strain, containing the indigenous plasmid pMEA300. No such mutants could be obtained in a strain devoid of pMEA300 sequences. Finally, chapters 8-9 summarize the results obtained, present concluding remarks and suggestions for further research.   10 

Biochemical and functional characterization of Nudix hydrolase enzymes with novel regulatory roles in Gram positive methylotrophic bacteria

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Biochemical and functional characterization of Nudix hydrolase enzymes with novel regulatory roles in Gram positive methylotrophic bacteria

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