A multitude of microorganisms possess the ability to metabolise molecular hydrogen (H2). The major enzyme family involved in hydrogen metabolism are Hydrogenases. These enzymes catalyse the reversible conversion of molecular hydrogen to protons and electrons (H2 ↔ 2H+ + 2e-). These enzymes have the potential to be utilised for biotechnological applications such as hydrogen fuel cells, but they also represent promising drug targets for inhibition of bacterial energy metabolism both within the gastrointestinal tract and after infection. Therefore, further understanding and discoveries made in the hydrogenase field warrants progression into applied medical and biotechnological research areas. Hydrogenases are also interesting due to their phylogeny and physiology in a large number of microbial species. These enzymes are categorised by their active site architecture. One well studied, ancient group is termed the [NiFe]-hydrogenases, which all harbour a complex NiFe(CN-)2CO active site in the 'large' catalytic subunit and usually have three iron-sulfur clusters within a 'small' electron transferring partner subunit. [NiFe]-hydrogenases have undergone massive diversification, with four major phylogenetic subgroups arising. The major part of this Thesis concerns work on a Group 4 [NiFe]-hydrogenase that functions in partnership with a formate dehydrogenase as a formate hydrogenlyase (FHL). This FHL complex generates H2 and CO2 from the disproportionation of formate (CHOO- + H+ ↔ H2 + CO2). In this Thesis, genetic and biochemical characterisation of Pectobacterium atrosepticum SCRI1043, a potato pathogen, led to the identification of a novel FHL complex. The [NiFe]-hydrogenase in this organism is similar to that of Escherichia coli Hydrogenase-4, with an extended membrane domain similar to that of respiratory Complex I. Importantly, the P. atrosepticum formate dehydrogenase is selenium-free, while previously characterised FHL complexes have selenocysteine-containing formate dehydrogenases. Using genetic and biochemical approaches it was shown that the [NiFe]-hydrogenase and a formate dehydrogenase were vital for H2 production by P. atrosepticum. Using plant infection assays it was also shown that the gene encoding the formate dehydrogenase was important for full infective ability of P. atrosepticum in potato plants and tubers. The latter part of this Thesis focuses on developing genetic tools to study this novel FHL from P. atrosepticum as well as Hydrogenase-1 and -2 from E. coli.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21132号 / 農博第2258号 / 新制||農||1056(附属図書館) / 学位論文||H30||N5106(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 小川 順, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

Arthrobacter sp. yra dirvoje paplitusios bakterijos, kurios gali prisitaikyti prie nepalankių aplinkos sąlygų, tokių kaip osmosinis šokas ar maisto medžiagų trūkumas. Osmoreguliacijoje daugelis organizmų naudoja glicino betainą. Yra žinoma, kad Arthrobacter globiformis bakterijos gali panaudoti glicino betainą kaip anglies ir azoto šaltinį, tačiau nebuvo aišku, ar Arthrobacter globiformis gali šią medžiagą naudoti kaip osmoprotektorių. Iš Arthrobacter globiformis buvo klonuotas DNR fragmentas, kuriame buvo identifikuoti genai, kurie tiesiogiai ir netiesiogiai dalyvauja glicino betaino katabolizme. Struktūrinė genų organizacija leidžia manyti, kad šios bakterijos gali panaudoti glicino betainą kaip osmoprotektorių, Taip pat buvo įdomu patikrinti, ar tokią funkciją glicino betainas gali vykdyti ir kitose A. globiformis giminingose bakterijose. Tyrimui buvo pasirinkti tipiniai Arthrobacter genties bakterijų kamienai (A. atrocyaneus, A. citreus, A. crystalopoietes, A. globiformis, A. ramosus, A. sulfureus bei Arthrobacter spp. (KA3, P3, KA2V2, PY22, KA2, GAZ21, P2G, KA4, KA2V3, GAZ3, PRH1, PY21, VM22, VP23, RD1, VM22, VP22, VP3, VPW7, VPS4, 96, 94, 85, 68M, 83 68B, BL-3 ir 1-IN). Darbo metu paaiškėjo, kad šį junginį apsaugai nuo osmosinio streso naudoja tik dalis Arthrobacter genties bakterijų. Glicino betainas artrobakterėse dažniau naudojamas kaip anglies šaltinis. / Arthrobacter spp. are wide-spread soil bacteria, which are adapted to grow under osmotic stress and shortage in available carbon (energy) sources. Many organisms utilise glycine betaine as an osmoprotectant. It is known, that Arthrobacter globiformis can use glycine betaine as a sole carbon and nitrogen source, however, it is unclear whether this bacterium use this substrate as osmoprotectant. A DNA fragment harbouring genes, which directly and indirectly are involved in glycine betaine degradation, was cloned from A. globiformis. The structural organization of these genes suggested, that this bacterium could use glycine betaine as an osmoprotectant. So, it was tested if other Arthrobacter spp. strains use glycine betaine to protect from osmotic stress. Typical Arthrobacter sp. strains (A. atrocyaneus, A. citreus, A. crystalopoietes, A. globiformis, A. ramosus, A. sulfureus and Arthrobacter spp. (KA3, P3, KA2V2, PY22, KA2, GAZ21, P2G, KA4, KA2V3, GAZ3, PRH1, PY21, VM22, VP23, RD1, VM22, VP22, VP3, VPW7, VPS4, 96, 94, 85, 68M, 83 68B, BL-3 and 1-IN) were chosen in this study. It turned out, that less than a half of studied strains use glycine betaine as osmoprotectant. The arthrobacters used glycine betaine more as a sole carbon source than as an osmoprotectant.

No description available.

Les déshydrogénases sont faiblement actives en présence de fortes concentrations (> 50 % (v/v)) de liquides ioniques (LIs) miscible à l’eau et les raisons précises de cette inactivation ne sont pas connues. La structure de la formiate déshydrogénase de Candida boidinii (FDH) en présence de ces LIs miscibles à l’eau a été étudiée par atténuation de fluorescence par de l’iode ou de l’acrylamide. Une concentration critique, la CILc ("Critical Ionic Liquid concentration"), au dessus de laquelle la fluorescence n’est pas exploitable, est déterminée. Elle se situe entre 30 et 40 % (v/v) des LIs de cette étude. Les LIs s’avèrent être de forts agents dénaturants : les constantes d’atténuation de fluorescence en présence de LI sont de 1,4 à 2 fois plus importantes qu’en présence d’urée. La FDH a été modifiée chimiquement par des cations analogues aux cations des LIs afin de la préserver de l’interaction avec les LIs. Les enzymes modifiées par des cations de plus petite taille présentent une importante activité résiduelle (30-45%) en présence de 70% (v/v) de LI tandis que l’enzyme sauvage est inactive. En présence de 30% (v/v) de LI, l’efficacité catalytique (kcat/KM) des enzymes modifiées augmente de 1,3 à 3,6 fois et la constante de Michaelis (KM) diminue de 1,7 à 4,6 fois suivant le cation utilisé. Selon la taille du cation greffé, le temps de demi-vie des enzymes modifiées augmente de 3 à 5 fois en solution tamponnée. Enfin, la structure des enzymes modifiées est préservée en présence de 40% (v/v) de LI tandis que l’enzyme native commence à se dénaturer. La technique d’évolution dirigée a également été utilisée. A ce jour, 987 mutants ont été criblés. Un mutant (M60) présente une activité résiduelle de 35% à 70% (v/v) de LI tandis que l’enzyme sauvage est inactive. Ce dernier porte deux mutations, N187S et T321S, situées à la surface de la structure protéique. En présence de 30 % (v/v) de LI, le mutant 60 fixe les substrats avec des valeurs de de KMNAD et de KDN3 (N3 est l’azide, un inhibiteur compétitif du formiate) 2 fois plus faibles que celles de l’enzyme sauvage. / The dehydrogenases are weakly active in the presence of high concentration (> 50% (v/v)) of water-miscible ionic liquids (ILs) and the mechanism of enzyme inactivation in ILs is not fully understood. The structure of the formate dehydrogenase Candida boidinii (FDH) in ILs has been studied by quenching of fluorescence experiments in the presence of iodide or acrylamide. A critical concentration, the CILc (“Critical Ionic liquid concentration"), is defined as the concentration of IL above which the fluorescence is not relevant. In this work, the CILc is comprised between 30 and 40 % (v/v) of the ILs. The ILs have revealed to be denaturing agents which increase the quenching of fluorescence efficiency by 1.4 to 2 times more than in urea. The FDH is chemically modified by analogous cations found in ILs in order to preserve the biocatalyst from the direct interaction with ILs. The enzymes modified by the smaller cations shown 30 45% residual activity at 70% (v/v) of ILs while the native enzyme is fully inactive. In the presence of 30% (v/v) of ILs, the kinetic efficiency (kcat/KM) of the grafted enzymes is improved by a 1.3-3.6 fold factor and the constant of Michaelis (KM) is reduced by a 1.7-4.6 fold factor depending on the grafted cations. In aqueous solution, the half-lives of modified enzymes are 3 to 5 fold higher than the native FDH depending on the size of the cation grafted. Finally, the structure of the grafted enzymes is somehow maintained in the presence of 40% (v/v) of ILs while the native FDH begins to unfold. We also used directed evolution to improve the FDH activity in the presence of ILs. At this stage, 987 mutants have been screened and one mutant (M60) shows 35% of residual activity at 70% (v/v) of ILs. In the presence of 30% (v/v) of ILs, the mutant binds the substrates in a greater extent than the native FDH, the values of KMNAD and the KDN3 (N3 is the azide, a competitive inhibitor of formate) are reduced by a 2 fold factor by comparison to the native enzyme.