Rhodobacter capsulatus is a photosynthetic, purple non-sulfur (PNS) bacterium that produces biohydrogen via photofermentation. Nitrogenase enzyme is responsible for hydrogen production / during fixation of molecular nitrogen into ammonium, hydrogen is produced. Since this process is an energetically expensive process for the cell, hydrogen production is strictly controlled at different levels. When ammonium is present in the environment, hydrogen production completely ceases. The key proteins in the regulation of nitrogenase by ammonium are two PII proteins / GlnB and GlnK. &lsquo / Hyvolution&rsquo / , 6th framework EU project, aims to achieve maximum hydrogen production by combining two hydrogen production processes / dark fermentation and photofermentation. In the first stage of the overall process, biomass is used for hydrogen production in dark fermentation process. Then, the effluent of dark fermentation is further utilized by photosynthetic bacteria to produce more hydrogen. However, the effluent of dark fermentation contains high amount of ammonium, which inhibits photofermentative hydrogen production. In order to achieve maximum hydrogen production, ammonium regulation of nitrogenase enzyme in R.capsulatus has to be released. For this purpose, all PII signal transduction proteins of R.capsulatus (GlnB and GlnK) were targeted to be inactivated by site-directed mutagenesis. The internal parts of glnB and glnK genes were deleted individually without using antibiotic cassette insertion. The successful glnB mutant was obtained at the end of mutagenesis studies. In the case of glnK mutation, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained. The effect of ammonium on glnB mutant R.capsulatus was investigated and compared with wild type. Biomass of the bacterial cultures, pH of the medium and amount of produced hydrogen were periodically determined. Moreover, the concentrations of acetic, lactic, formic and propionic acids in the medium were periodically measured. Both wild type and glnB mutant grew on acetate and effectively utilized acetate. Ammonium negatively affected hydrogen production of glnB mutant and wild type. The ammonium inhibition of hydrogen production did not release in glnB mutant due to the presence of active GlnK protein in the cell / hence, inactivation of one of PII proteins was not enough to disrupt ammonium regulation of the cell. Moreover, kinetic analysis of bacterial growth and hydrogen production were done. Growth data fitted to the Logistic Model and hydrogen production data fitted to the Modified Gompertz Model.

Genomic and proteomic investigations have yielded, and continue to produce, a large amount of information about genes and their protein products. In contrast, the evidence bearing on physiological roles of specific proteins is much more scarce. To address the functional part of biological inquiry, one would like to perturb, at will and selectively, the function of any protein of interest in vivo and to analyze the resulting phenotypic effects, thereby probing the protein’s role in a cell. Ideally, a method for doing so should be applicable both to individual gene products and to a large collection of them. Gene knockouts, a powerful tool to study gene function, have limitations in the study of development when the early phenotypes are cell- or organismal- lethal. Conditional mutants are particularly useful for analysis of genes whose functions are essential for the organism’s viability. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and shows an inactive phenotype under a different set of conditions, called nonpermissive; the latter must be still permissive for the wild type (wt) allele of a gene. Conditional mutants make possible the analysis of physiological changes that follow controlled inactivation of a gene or gene product and can be used to address the function of any gene. Temperature sensitive (ts) mutants are an important class of conditional mutants whose phenotype is similar to that of wt at lower (permissive) temperature, but show low or reduced level of activity above a certain temperature called restrictive temperature, while the wt gene shows a similar phenotype at both the temperatures. Ts mutants provide an extremely powerful tool to study gene expression in vivo and in cell culture. They provide a reversible mechanism to lower the level of a specific gene product simply by changing the temperature of growth of the organism. Ts mutants are typically generated by random mutagenesis; either by ultraviolet light, a chemical mutagen or by error-prone PCR followed by often laborious screening procedures. Therefore, they are cumbersome to make, especially in the case of organisms with long generation times. Keeping in view the importance of ts mutants in biology, Varadarajan et al. 1996, had developed an algorithm to predict ts mutants at predicted, buried sites of a globular protein from its amino acid sequence. Experimental tests of the algorithm were carried out on the CcdB toxin of Escherichia coli to further refine and improve the method (Chakshusmathi et al. 2004). Based on this result simple rules for the design of ts mutants were suggested. This thesis aims at validating and improving on these rules and to find out if ts mutants of a protein can also be generated by perturbing functionally important residues. In addition, it is currently unclear with what frequency ts mutants of a protein isolated in one organism will show a ts phenotype in a completely different organism. This thesis makes preliminary efforts to address this issue. The model system chosen to carry out these studies is a protein called Gal4, which is a yeast transcriptional activator. This protein is biologically relevant as it has been used for ectopic gene expression in diverse organisms including yeast, fruitflies, zebrafish, mice and frogs (Ornitz et al. 1991; Brand and Perrimon 1993; Rahner et al. 1996; Andrulis et al. 1998; Scheer and Camnos-Ortega 1999; Hartley et al. 2002). The introductory chapter (Chapter 1) discusses the importance of ts mutants and our understanding and progress in this field so far, relevant for the work reported in this thesis. Chapter 2 describes generation of ts mutants of Gal4 in yeast. Full length Gal4 (fGal4) is an 881-aa protein. To simplify the construction of ts Gal4, we have designed a functional truncated Gal4 (miniGal4 or mGal4) of 197 residues. Five residues (9, 10, 15, 18 and 23) of the Gal4 DNA binding domain, which are in close contact with the DNA, were randomized in mGal4. Based on average hydrophobicity and hydrophobic moment, 68, 69, 70, 71, and 80 are the only residues in the region 1-150 that are predicted to be buried at the 90% confidence level. Of these five sites, residues 68, 69 and 70 were chosen for mutagenesis. At these three sites, four stereochemically diverse substitutions (Lys, Ser, Ala and Trp) were made. In a separate set of experiments each predicted, buried residues were also individually randomized in both mini and in full length Gal4 (fGal4). In all cases, we have been successful in isolating ts mutants in more than one position. At both permissive and restrictive temperatures, the activity of the Gal4 ts mutants is substantially lower than the wt. However, at the restrictive temperature, the activity of the ts Gal4 is lowered below the threshold required for reporter gene expression. This view of how ts mutants function is quite different from the general notion that the ts and wt behave similarly at permissive temperatures. Chapter 3 deals with transferability of two of the ts constructs mutated at DNA binding residues (R15W and K23P) to Drosophila. Two fGal4 encoding DNA fragments carrying the mutations were cloned into P element vectors under control of Elav and GMR promoters and several transgenic Drosophila lines were generated. These were crossed to various UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. We show that both of these yeast ts mutants also show a ts phenotype in Drosophila. We have compared our ts Gal4 system with a popularly used system (TARGET) (McGuire et al. 2003) used for conditional gene expression in Drosophila. Our ts Gal4 mutants appear to provide tighter control at the restrictive temperature and a more uniform and rapid induction of gene expression upon shifting from the restrictive to the permissive temperature than the TARGET system with the promoters and the reporters we have used. Although cold sensitive (cs) mutants are often more useful than ts mutants, for reasons currently unclear, cs mutants are much more difficult to isolate than ts mutants. In Chapter 4, we have attempted to convert the ts phenotypes observed with Gal4 mutants in Drosophila and CcdB mutants in E. coli (Chakshusmathi et al. 2004) to cs phenotypes by increasing the expression level of these mutant proteins selectively at higher temperature. Several ts mutants of CcdB have been previously reported (Chakshusmathi et al. 2004). For converting the ts phenotype observed by E. coli toxin CcdB mutants (Chakshusmathi et al. 2004) to a cs phenotype, the arabinose inducible plasmid pBAD24CcdB and its mutant derivatives were used. By inducing expression of the mutant protein at higher temperature with arabinose, while keeping the basal level of expression without arabinose at lower temperature, we have been able to show cold sensitive behavior by these CcdB ts mutants in E. coli. For producing a cs phenotype with Gal4 mutants in Drosophila, we have used a P element vector where the GMR element is placed in-between hsp70 binding sites. This driver results in enhanced expression of downstream genes at 30 relative to 18°C because of the presence of the hsp elements (Kramer and Staveley 2003). Ts mutants at DNA binding and buried residues of fGal4 were cloned into this vector and several transgenic lines for each construct were obtained. The Gal4 mutants at exposed DNA binding residues but not at buried residues show a cs phonotype when they were crossed to various UAS-reporters lines. The buried residue mutants are likely to be destabilized and their degradation pathway might differ in yeast and in Drosophila. Because of this, these mutants might not be showing the desired cs phenotype in Drosophila. Although mGal4 and fGal4 have very similar activities in yeast, it was necessary to examine if they also had identical activities in Drosophila. Determining their relative activities in Drosophila is the aim of Chapter 5. To this end, mGal4 was cloned into P element vectors under control of hsp70 or GMRhs promoters and transgenic flies were generated. The transgenic lines were crossed to various UAS-reporters and reporter gene activities in the progeny were characterized. Although mGal4 and fGal4 showed similar activity in yeast, in Drosophila for reasons that are currently unclear, mGal4 was considerably less active than fGal4. As some of the fGal4 mutants showed a cs phenotype under GMRhs driver as shown in the earlier chapter (Chapter 4), several ts mutants of mGal4 in yeast in buried and as well as at the DNA binding residues were transferred to Drosophila under hs and GMRhs promoter. The transgenic lines obtained were tested for cold sensitivity by crossing with various UAS-reporter lines. However, in all cases mutant mGal4 showed an inactive phenotype in Drosophila. We suggest that this is because the intrinsic activity of these mGal4 mutants is substantially weaker than wt mGal4 even at permissive temperature in yeast. The further lowering of activity in Drosophila pushes the activity below the threshold required for reporter gene expression resulting in an inactive phenotype. The concluding chapter (Chapter 6) summarizes the conclusions drawn from this entire study and provides insights into possible mechanisms responsible for ts and cs phenotypes. The mutant phenotypes of Gal4 in yeast and in Drosophila suggest that ts phenotypes appear to result from a threshold effect. Such mutations lower the activity and/or level of the protein relative to the wt at all temperatures. Since maximal stability temperatures are rarely in excess of room temperature, with an increase in temperature, the activity of an already marginally active mutant can fall below the threshold required for function resulting in a temperature sensitive phenotype. The strategies we used for producing ts mutants have several advantages over standard approaches of generating ts alleles by random mutagenesis. We anticipate that conclusions of this study would be useful for generation of ts mutants of other globular proteins in diverse organisms. We also show that exposed, functional residues involved in protein: ligand or protein: protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms. In addition, the active site mutants of fGal4 in Drosophila, which show ts and cs phenotypes depending on the Drosophila promoter chosen for expression, can be used for conditional and reversible expression of a number of other genes using the Gal4-UAS system (Brand and Perrimon 1993).

<p>Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity.</p><p>In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions.</p><p><b>Key words:</b>Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity</p>

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 114-126.

In mature chloroplasts, the DNA (cpDNA) is surrounded by a potentially genotoxic environment that would make the mitochondrial DNA milieu look like a “nadree” (picnic). And yet, the slower evolution of cpDNA compared to other cellular genomes suggests that this organelle must have efficient mechanisms for repairing DNA. Unfortunately, those mechanisms have been barely noted, much less studied. This dissertation describes a novel approach that was developed to study how chloroplasts of Arabidopsis repair the most severe form of DNA damage, a double-strand break (described in Chapter 2). The success with this approach also prompted the development of a new method for site-specific modification of tobacco cpDNA that is described in Chapter 3. To study the consequences and repair of a break in the circular plastid genome, we developed an inducible system based on a psbA-intron endonuclease from Chlamydomonas (I-CreII) that specifically cleaves the psbA gene of Arabidopsis. The protein was targeted to the chloroplast using the rbcS1 transit peptide, and activation of the nuclear gene was made dependent on an exogenous inducer (β-estradiol). In Chlamydomonas, I-CreII cleavage at psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) that are abundant in that genome. By comparison, Arabidopsis cpDNA is very repeat-poor. Nonetheless, phenotypically strong and weak transgenic lines were obtained, and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of psbA, but not cpDNA as a whole, in the strongly-expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site that were indicative of repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) or no homology. The results provide evidence of alternative repair pathways in the Arabidopsis chloroplast that resemble the nuclear microhomology-mediated and nonhomologous end-joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, plus other considerations, the data suggests that an evolutionary relationship may exist between the repeat structure of cpDNA and the organelle’s ability to repair broken chromosomes. Taking advantage of the inducible I-CreII system, I developed a method to delete defined regions of cpDNA in tobacco, which was named DREEM (for direct repeat and endonuclease mediated). Chloroplast transformation was used to introduce an I-CreII cleavage site adjacent to an aadA:gfp marker and flanked by a direct repeat of 84 bp. When chloroplast-targeted I-CreII was induced with β-estradiol during germination, complete loss of the aadA:gfp marker occurred by SSA-type repair involving the 84-bp direct repeat. I obtained additional evidence for DREEM effectiveness by deleting 3.5 kb of native cpDNA that included part of the large ycf1 gene. DREEM can be used for other modifications besides gene deletions, partly because it is seamless and leaves no trace of introduced DNA. Since expression of the endonuclease is controlled by steroid application (and concentration), and the deleted cpDNA is probably destroyed during the SSA process, this inducible gene-ablation technique could enable the study of essential chloroplast genes in vivo. / text

If small isoprenoids, the starting component of carotenoids, can be efficiently excreted from thermophilic cyanobacteria, they could help satisfy the demand for sustainably produced hydrocarbons. This is the driving force behind wanting to understand the response and regulation of isoprenoid pathways to environmental stimuli in the thermophilic cyanobacterium, Thermosynechococcus elongatus, BP-1. The portion of the isoprenoid pathway studied here is the carotenoid pathway since these products are critical to adaptation and they encompass the largest pool of isoprenoid compounds in cyanobacteria. Although synthetic biology in cyanboacteria has improved in recent years, there are many undiscovered metabolic complexities that make large-scale commercial production challenging. To address this need, I quantify and report for the first time metabolic shifts within the carotenoid pathway of BP-1 due to combined effects of temperature, pH and blue light. I show that metabolism shifts from the dicyclic into the monocyclic carotenoid pathway in response to pH, and that decreasing temperature drives flux into the end products of both pathways. Also, I report that the productivity of an uncommon carotenoid, 2-hydroxymyxol 2’-fucoside (HMF), approached 500 μg/L-day in cultures grown at 45 °C, high light intensity, and pH 8. In order to further elucidate these responses, I analyzed 42 RNAseq samples taken over time of BP-1 induced by cold and heat stress and compared these results to metabolomics data. I showed that crtR and crtG, two central carotenogenesis genes, are transcriptionally controlled and used weighted gene co-expression network analysis (WGCNA) to determine eight separate co-expressed modules of biological significance. Among the co-regulated heat response and cold response genes there were three and five non-coding RNA, respectively, providing targets for future investigation. Using subtractive genomics and transcriptional data I narrowed the potential missing steps of the myxol pathway in cyanobacteria to seven unknown BP-1 genes, two of which were confirmed not to be involved in the missing step(s). Finally, by generating a ΔcrtG mutant and testing it under different environmental parameters, I showed that HMF does not protect against high pH or low temperature (despite up-regulation at these conditions), and that CrtG has a higher affinity for monocyclic than dicyclic carotenoids. / text

Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine. / text

Ferrochelatase catalyzes the terminal step of heme biosynthesis by inserting ferrous iron into protoporphyrin IX. The current study is aimed at understanding the structural basis of porphyrin binding and distortion in ferrochelatase-catalyzed reaction by functional analysis of a highly conserved active site loop motif. The loop was shown to contact bound porphyrin based on crystallographic and molecular modelling observations, and its role in murine ferrochelatase was assessed by random mutagenesis and steady-state kinetic analysis. To overcome the limitations of conventional kinetic assay methods for ferrochelatase, a continuous assay was developed by monitoring porphyrin fluorescence decrease using natural substrates ferrous iron and protoporphyrin IX under anaerobic conditions. For wild-type murine ferrochelatase, the assay yielded KmPPIX of 1.4 uÌ?M, KmFe2+ of 1.9 uÌ?M and kcat of 4.0 min-1 at 30 °C. The results of random mutagenesis indicated that all the loop re sidues spanning Q248-L257 tolerated functional substitutions. While Q248, S249, G252, W256 and L257 possessed high informational content, the other five positions contained low informational content. Selected active loop variants exhibited kcat comparable to or higher than that of wild-type enzyme, while KmPPIX was increased in most variants. The kcat/KmPPIX remained largely unchanged, with the exception of a 10-fold reduction in variant K250M/V251L/W256Y. Molecular modeling of the active loop variants suggested that loop mutations resulted in alterations of the active site architecture. Distortion of porphyrin substrate, a crucial step in ferrochelatase-catalyzed metal chelation, was examined using resonance Raman spectroscopy. The results revealed that both wild-type enzyme and loop variants induced saddling of substrate protoporphyrin. Further, loop mutations generally interfered with porphyrin saddling, with the least deformation observed in variant K250M/V251L/W256Y.N-alkyl porphyrins are potent competitive inhibitors of mammalian ferrochelatase. The present study showed that while N-methyl protoporphyrin strongly inhibited the wild-type enzyme with an inhibition constant in the nanomolar range, it was less effective in inhibiting variants P255R and P255G. These results suggest that inhibitor binding may be associated with a protein conformational change mediated by P255. Wild-type ferrochelatase is a homodimeric [2Fe-2S] cluster-containing protein. Variants S249A/K250Q/V251C and S249A/K250R/G252W were found to retain enzymatic activity in the absence of FeS cluster and form active, higher order oligomers. These observations raise the possibility that FeS cluster and homodimeric organization are not essential to ferrochelatase catalysis.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19069号 / 人博第722号 / 新制||人||173 / 32020 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 宮下 英明, 教授 川本 卓男 / 学位規則第4条第1項該当

Lipoxygenases (LOX) catalyze regio- and stereospecific oxygenation of polyunsaturated fatty acids to hydroperoxides. These hydroperoxides are further metabolized to leukotrienes and lipoxins in mammals, and are involved in asthma and inflammation. LOX of animals and plants contain iron as catalytic metal (FeLOX). Filamentous fungi use both FeLOX, and manganese containing LOX (MnLOX). The role of LOX in fungi is still not known. This thesis focuses on expression of novel MnLOX, analyses of their reaction mechanism and products by HPLC-MS/MS, protein crystallization and analysis of the first MnLOX structure.   MnLOX from G. graminis, M. salvinii, M. oryzae, F. oxysporum and C. gloeosporioides were expressed in Pichia pastoris, purified and characterized by HPLC-MS/MS. All MnLOX catalyzes suprafacial hydrogen abstraction and oxygen insertion. Replacement of one Ile to Phe in the active site of MnLOX of G. graminis could switch the mechanism from suprafacial to mainly antarafacial. MnLOX of F. oxysporum was interesting since it catalyzes oxygenation of linoleic acid to 11R- instead of the more common 11S-hydroperoxides. This feature could be attributed to a single Ser/Phe exchange in the active site.   We found that Gg-MnLOX utilizes hydrogen tunneling in the reaction mechanism, but was slightly more temperature dependent than soybean FeLOX. It is an intriguing question why some fungal LOX use manganese and not iron as catalytic metal and whether the large redox potential of Mn2+/Mn3+ (1.5 V) can be tuned close to that of Fe2+/Fe3+ (0.77 V) for redox cycling and catalysis. We present crystallization conditions for two MnLOX, and the 2.07 Å crystal structure of MnLOX from M. oryzae, solved using sulfur and manganese single anomalous dispersion (SAD). The structure reveals a similar metal coordinating sphere as FeLOX but the metal ligand Asn473 was positioned on a short loop instead of a helix and formed interactions with a conserved Gln. This feature could be essential for the use of manganese as catalytic metal in LOX. We found three Phe residues that likely facilitate the suprafacial hydrogen abstraction and oxygen insertion for MnLOX. These findings provide new insight into the unique reaction mechanism of MnLOX.