Phenylketonuria (PKU) and related forms of non-PKU hyperphenylalaninemias (HPA) result from deficiencies in phenylalanine hydroxylase (PAH), the hepatic enzyme that catalyses the conversion of phenylalanine (phe) to tyrosine (tyr). Patients are characterised by a metabolic phenotype comprising elevated levels of phe and some of its metabolites, notably phenyllactate (PLA), phenylacetate (PAA) and phenylpyruvate (PPA), in both tissue and body fluids. Treatment from birth with low-phe diet largely prevents the severe mental retardation that is its major consequence. / Mechanisms underlying the pathophysiology of PKU are still not fully understood; to this end, the availability of an orthologous animal model is relevant. A number of N-ethyl-N-nitrosourea (ENU) mutagenized mouse strains have become available. I report a new heteorallelic strain, developed by crossing female ENU1 (with mild non-PKU HPA) with a male ENU2/+ carrier of a 'severe' PKU-causing allele. I describe the new hybrid ENU1/2 strain and compare it with control (BTBR/Pas), ENU1, ENU2 and the heterozygous counterparts. The ENU1, ENU1/2 and ENU2 strains display mild, moderate and severe phenotypes, respectively, relative to the control and heterozygous counterparts. / I describe a novel method using negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS) to measure the concentration of PLA, PAA and PPA in the brain of normal and mutant mice. Although elevated moderately in HPA and more so in PKU mice, concentrations of these metabolites are not sufficient to explain impaired brain function; however phe is present in brain at levels associated with harm. / Finally, I describe a new modality for treatment of HPA, compatible with better human compliance: it involves enzyme substitution with non-absorbable and protected phenylalanine ammonia lyase (PAL) in the intestinal lumen, to convert L-phenylalanine to the harmless metabolites (trans-cinnamic acid and trace ammonia). PAL, taken orally, substitutes for the deficient PAH enzyme and depletes body pools of excess phe. I describe an efficient recombinant approach to produce PAL enzyme. I also provide proofs of both pharmacologic and physiologic principles by testing PAL in the orthologous mutant mouse strains with HPA. The findings encourage further development of PAL for oral use as an ancillary treatment of human PKU.

Cell wall loosening and degradation are important processes in major stages of plant development including fruit ripening. Three main mechanisms have been proposed to contribute towards cell wall polysaccharide degradation in vivo: enzymic hydrolysis by endopolygalacturonase (EPG), enzymic elimination by pectate lyase (PL), and non-enzymic scission by hydroxyl radicals (•OH). However, little idea as to which of these three mechanisms predominates in homogalacturonan degradation especially during fruit ripening. This study presents an attempt to discover the respective contribution of those three mechanisms of attack. The strategy used to achieve the objective of this study was to identify and measure homogalacturonan molecules that exhibit symptoms of each mechanism of attack. A method that was developed in this study is a fluorescent labelling method mainly to study the •OH attack on pectic polysaccharides. This labelling method is based on the ability of 2-aminoacridone (2-AMAC) to reductively aminate oxo groups of sugar moieties followed by exhaustive digestion with Driselase. In a model in-vitro experiment, the developed novel fluorescent labelling method, when applied to homogalacturonan, that had been attacked by •OH (Fenton reagent), produced at least three fluorescent ‘fingerprint’ compounds, separable by high-voltage paper electrophoresis (HVPE) based on their charge/mass properties at pH 6.5 and also by high pressure liquid chromatography (HPLC) on a C18 column with a fluorescence detector at λem= 520 nm. These fingerprint compounds include: a monomer, 1A*; a dimer, 2A*; and an unidentified compound, X*. In-vivo application with alcoholinsoluble residue (AIR) of seven species of fruit (pear, mango, banana, apple, avocado, strawberry and strawberry tree fruit) at three stages of softening produced at least two fluorescent fingerprint compounds: a monomer, 1AF and a dimer, 2AF. XF, an interesting compound found in a few samples in in-vivo experiments, showed electrophoretic mobility similar to X*; however, the retention time of this compound on HPLC did not agree with that of X*. 2AF was suggested to be exclusive evidence for •OH attack in vivo while 1AF was suggested to be a useful evidence not only to reveal •OH attack but also to reveal EPG and PL attack on pectic polysaccharides during fruit softening. HVPE and HPLC results showed an increasing pattern of 2AF in mango, banana, avocado and strawberry tree fruit, which indicated progressive •OH attack on pectic polysaccharides during the softening process. There was no clear evidence of 2AF at any stage of softening in apple and strawberry, which may suggest that fruit softening in apple and strawberry was not associated with •OH attack. On the other hand, HVPE analysis of 1AF showed and increasing pattern in pear, mango, banana, avocado and strawberry tree fruit, which may indicate EPG, PL and/or •OH attack during fruit softening. Production of these fluorescent fingerprint compounds provides good evidence for •OH attack on pectic polysaccharides, and has the potential to give useful information for EPG and PL attack in vivo.

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine. Mechanistic characterization of ADE from Escherichia coli was performed along with biophysical studies. The structure of ADE was solved from A. tumefaciens. The structure, along with the biochemical and biophysical characterization, enabled the elucidation of the mechanism of the deaminase reaction of ADE. Elucidation of the origin of the oxygenation reactions within ADE led to the discovery of a promiscuous catalase reaction. The diiron ADE from all tested bacterial species exhibited this unusual reaction, along with the generation of superoxide and hydroxyl radicals, the latter being responsible for the oxygenation of the protein. The residues that were identified to be oxygenated were primarily the metal binding residues implying the origin of this reaction was the binuclear iron center. A group of bacterial enzymes that are co-localized in the same genomic operon as ADE but of unknown function were identified. The enzyme Bh0637 from Bacillus halodurans, a representative member of this group of enzymes was characterized. This enzyme was shown to preferentially catalyze the deamination of epigenetic base, N-6-methyadenine. Lastly, gram-negative bacteria have a highly conserved phn operon composed of 14 genes to break the C-P bond of inert alkylphosphonates. The genes phnGHIJKLM are absolutely critical for this activity. We discovered that methylphosphonate reacts first with MgATP to form alpha-D-ribose-1-methylphosphonate-5-triphosphate (RPnTP) and adenine by the action of PhnI, PhnG, PhnH and PhnL. PhnI by itself was shown to perform a novel nucleosidase reaction converting MgATP to ribose-5-triphosphate and adenine. The triphosphate moiety of RPnTP is then hydrolyzed to pyrophosphate and alpha-D-ribose-1-methylphosphonate-5-phosphate (PRPn) by PhnM. The carbon-phosphorus bond of PRPn is subsequently cleaved via a radical-based reaction to alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate (PRcP) and methane in the presence of S-adenosyl-L-methionine by PhnJ.

Phenylketonuria and hyperphenylalanemia are autosomal recessive inborn errors of phenylalanine metabolism that are caused by mutations in the phenylalanine hydroxylase gene. Due to the stringency of the present dietary therapy, alternative treatments are being studied. Phenylalanine ammonia-lyase (PAL) is one of the potential dietary supplements for these patients. PAL is a well-studied plant enzyme which breaks down phenylalanine into trans-cinnamic acid and ammonia (Camm and Towers, 1973). It is found in the cytoplasm of the plant cells and is naturally encapsulated by plant cell walls which may protect it against the acidic pH environment in the gastrointestinal tract. It presumably degrades ingested Phe that circulates in the intestinal lumen. In this study, red spring wheat seedlings (Triticum aestivum L.) found to contain high PAL activity naturally were investigated as a potential alternative oral therapy. Specifically, the objectives were (1) to evaluate different drying methods on generating concentrated and dried preparation of wheat seedlings containing high levels of PAL activity; (2) to examine the retention of PAL activity over three months of storage under various storage conditions; (3) to determine the stability of PAL activity in simulated human digestion condition to establish if further study of using plant source enzyme in vivo is warranted. Freeze-drying (FD) was found to have retained the most activity (>90 % recovery dry wt basis) compared to air-drying (AD) and vacuum-microwave drying (VMD) for both leaf and residual seed/root samples. Pre-freezing of leaf tissues at -18 °C before FD significantly retained the highest PAL activity compared to pre-freezing at -25 °C, -35 °C, and -80 °C (P<0.05). Over three months of storage, 60-80 % of PAL activity was recovered in leaf and —100 % was recovered in residual seed/root tissues after storage at -20 °C. After in vitro protein digestion, 36% and 42 % of PAL activity was recovered in fresh leaf and root tissues respectively; however, FD tissues were found to be susceptible to proteases and acidic environment and no activity was recovered after three hours of in vitro protein digestion. High performance liquid chromatography (HPLC) analysis of the residual Phe after in vitro protein digestion confirmed that fresh tissues had significantly higher conversion of Phe than that of FD tissues. Together, these results suggest that red spring wheat seedlings may have potential as a dietary supplement for phenylketonuric patients while further study to enhance PAL activity in plant preparations is required.

Universiẗat, Diss., 2000--Kiel.

The three-dimensional structures of biological macromolecules and molecular assemblies are becoming increasingly important with the changing methodologies of drug discovery. The structures aid in understanding of protein function at the molecular level: be it a macromolecular assembly, a cytosolic enzyme or an intermembrane receptor molecule. X-ray crystallography is the most powerful technique to obtain the three-dimensional structures of such molecules at or near atomic resolution. With such a wide-spread importance, crystallography is an integral part of structural biology and also of the current drug discovery programs. The present thesis mainly deals with application of the crystallographic techniques for understanding the structure and function of adenylosuccinate lyase (ASL) from bacterial pathogens Salmonella typhimurium and Mycobacterium tuberculosis as well as its non-pathogenic counterpart Mycobacterium smegmatis. Studies were also carried out to understand the structure-function relationship of the protease in the plant virus Sesbania Mosaic Virus (SeMV). The thesis has been divided into six chapters. The first chapter contains an introduction to nucleotide synthesis and ASL superfamily of enzymes known as the aspartase/fumarase superfamily based on the published literature. Chapter 2 provides the details of the techniques used for the investigations presented in this thesis. Chapters 3-5 deal with the structural and functional studies carried out on ASL from the three bacterial organisms. Chapter 6 deals with the simulation studies carried out on SeMV protease. Mechanism and importance of nucleotide synthesis is introduced in Chapter 1, with special emphasis on purine de novo and salvage pathways. ASL is introduced as an important enzyme for purine synthesis. Its superfamily, the aspartase/fumarase superfamily of enzymes is described in detail with respect to its structure, function and pathophysiology. Objectives of the present study are outlined towards the end of the chapter. The experimental and computational techniques utilized during the course of my research are described in Chapter 2. These techniques include gene cloning, protein expression and purification, kinetic and biophysical characterization of proteins, crystallization, X-ray diffraction, data collection and processing, structure solution, refinement, model building, validation and structural analysis, phylogenetic studies, molecular docking and molecular dynamic simulation studies. Adenylosuccinate lyase is an important enzyme participating in purine biosynthesis. With the emergence of drug resistant variants of various pathogens, ASL has been recognized as a drug target against microbial infections. Chapter 3 deals with the structural and functional characterization of ASL from Salmonella typhimurium. Two constructs of the StASL gene were cloned and expressed leading to the purification of truncated (residues 1-366) and full-length (residues 1-456) polypeptides. Crystallization of the two polypeptides resulted in three independent structures. The full-length structure was very similar to the E. coli ASL structure consistent with 95% amino acid sequence identity between the two polypeptides. However, the truncated structures showed large distortions, especially of the active site residues, accounting for the catalytic inactivity of the truncated polypeptide in spite of retaining all residues considered important for function. The full-length ASL was catalytically active. A unique feature observed in StASL, not reported in other ASLs, was its allosteric regulation by the substrate. Kinetic studies also revealed hysteretic behavior of the enzyme. The electron density map of the full-length structure showed two novel densities on the molecular 2-fold axis into each of which a molecule of cadavarine could be fitted. Docking studies revealed a ligand-binding site at the inter-subunit interface between the two observed densities which might represent a potential allosteric site. Combining the structural and kinetic results, a possible morpheein model of allosteric regulation of StASL was hypothesized. Chapter 4 deals with the crystallographic and kinetic investigations on ASL from Mycobacterium smegmatis and Mycobacterium tuberculosis. MsASL and MtbASL were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at 2.16 Å resolution. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. Most of the active site residues were found to be conserved with the exception of Ser 148 and Gly 319 of MsASL. Ser 148 is structurally equivalent to a threonine in most other ASLs. Gly 319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active when compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria, their high GC containing genomes as well as with their dependence on other salvage pathways for the supply of purine nucleotides. Chapter 5 deals with the identification of the catalytic residues important for ASL catalysis in view of the earlier conflicting reports on the identity of these residues. pH-dependent kinetic studies were performed on full-length StASL. The theory behind these studies is also described in this chapter. Two residues with pKa values of 6.6 and 7.7 were identified as essential for the enzymatic activity. These results were interpreted along with structural comparison of MsASL and other superfamily enzymes with ordered C3 loops. They suggest that His 149 and either Lys 285 or Ser 279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. The final Chapter 6 of the thesis deals with the structural and dynamic studies carried out on Sesbania mosaic virus (SeMV) protease. The chapter begins with a general introduction to viruses, followed by a brief summary of SeMV. The goal of this study is to understand the interactions between the protease and VPg at a structural level using the information available from biochemical studies. Crystallographic studies initiated for the mutant H275APro and Y315APro were unsuccessful due to the insolubility of the proteins. Co-crystallization or soaking experiments of wild type protease with cognate peptides were unsuccessful due to the inability of the enzyme to bind to its substrates in the absence of VPg. Higher resolution structure of wild type protease did not yield any new insights when compared to the earlier reported structure determined at a lower resolution. In the absence of structural insights, molecular dynamic simulations were carried out on wild type protease structure and in silico generated mutants using GROMACS package. The studies showed the importance of flipping of residue Phe 301 and opening-closing of the loop region corresponding to residues 301-308 for the catalytic mechanism. The thesis concludes with Future perspectives of the various studies carried out on ASL and SeMV protease. The atomic coordinates determined from the work presented in this thesis have been deposited in the PDB and the assigned PDB codes are reported in the respective chapters. Publications cited in the thesis are listed in the Bibliography section.

Phenylketonuria and hyperphenylalanemia are autosomal recessive inborn errors of phenylalanine metabolism that are caused by mutations in the phenylalanine hydroxylase gene. Due to the stringency of the present dietary therapy, alternative treatments are being studied. Phenylalanine ammonia-lyase (PAL) is one of the potential dietary supplements for these patients. PAL is a well-studied plant enzyme which breaks down phenylalanine into trans-cinnamic acid and ammonia (Camm and Towers, 1973). It is found in the cytoplasm of the plant cells and is naturally encapsulated by plant cell walls which may protect it against the acidic pH environment in the gastrointestinal tract. It presumably degrades ingested Phe that circulates in the intestinal lumen. In this study, red spring wheat seedlings (Triticum aestivum L.) found to contain high PAL activity naturally were investigated as a potential alternative oral therapy. Specifically, the objectives were (1) to evaluate different drying methods on generating concentrated and dried preparation of wheat seedlings containing high levels of PAL activity; (2) to examine the retention of PAL activity over three months of storage under various storage conditions; (3) to determine the stability of PAL activity in simulated human digestion condition to establish if further study of using plant source enzyme in vivo is warranted. Freeze-drying (FD) was found to have retained the most activity (>90 % recovery dry wt basis) compared to air-drying (AD) and vacuum-microwave drying (VMD) for both leaf and residual seed/root samples. Pre-freezing of leaf tissues at -18 °C before FD significantly retained the highest PAL activity compared to pre-freezing at -25 °C, -35 °C, and -80 °C (P<0.05). Over three months of storage, 60-80 % of PAL activity was recovered in leaf and —100 % was recovered in residual seed/root tissues after storage at -20 °C. After in vitro protein digestion, 36% and 42 % of PAL activity was recovered in fresh leaf and root tissues respectively; however, FD tissues were found to be susceptible to proteases and acidic environment and no activity was recovered after three hours of in vitro protein digestion. High performance liquid chromatography (HPLC) analysis of the residual Phe after in vitro protein digestion confirmed that fresh tissues had significantly higher conversion of Phe than that of FD tissues. Together, these results suggest that red spring wheat seedlings may have potential as a dietary supplement for phenylketonuric patients while further study to enhance PAL activity in plant preparations is required. / Land and Food Systems, Faculty of / Graduate

A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69 % A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18475号 / 農博第2075号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4859(農学部図書室) / 31353 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 保川 清, 准教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

L’hydroperoxyde lyase (HPL) est une enzyme issue de la voie de la lipoxygénase, voie métabolique très représentée chez les végétaux, impliquée dans la production de composés aromatisants (l’hexanal, le 3Z-hexenal et le 2E-hexenal). Ces composés sont responsables de l’odeur fraîche de l’herbe coupée dite « note verte » et sont très utilisés par les industries cosmétiques et agroalimentaires. Leur biosynthèse résulte de l’oxydation des acides gras polyinsaturés en hydroperoxydes par la lipoxygénase, puis de leur clivage par l’hydroperoxyde lyase (HPL). Les procédés actuels de production de ces composés présentent certains inconvénients, ils sont notamment très polluants et peu performants, aussi l’utilisation d’enzymes recombinantes dans de tels procédés permettrait d’obtenir ces molécules de manière plus efficace tout en bénéficiant du label "naturel". L’ADNc codant pour l’hydroperoxyde lyase (HPLwt) a été isolé au laboratoire à partir d’olives noires. Afin d’améliorer la solubilité de l’enzyme, une HPL dépourvue de son peptide de transit chloroplastique (HPLdel) a également été produite. Les deux enzymes ont été exprimées chez E.coli, purifiées par chromatographie d’affinité puis caractérisées biochimiquement. Elles agissent exclusivement sur les 13-hydroperoxydes (13-HPL) à un pH et une température optimum de 7,5 et 25°C. De plus l’évaluation des paramètres cinétiques de l’enzyme montre qu’elles ont une meilleure efficacité catalytique (kcat/Km) sur les 13-hydroperoxydes d’acide linolénique (3,68 s-1.µM-1) que sur les 13-hydroperoxydes d’acide linoléique (0,54 s-1.µM-1). La bioconversion des 13-hydroperoxydes d’acide linoléique et linolénique en hexanal et 3Z-hexénal par l’action de l’HPLwt et l’HPLdel a été étudiée. Des taux de conversion maximum atteignant 93 % et 68 % pour la production d’hexanal et 73 % et 45% pour la production d’3Z-hexénal ont été obtenus quand l’HPLwt et l’HPLdel sont utilisées respectivement. La stabilité de l’enzyme a ensuite été étudiée. Des essais de conservation montrent que l’utilisation de glycérol à 10% (v/v) permet le maintien de la totalité de l’activité de l’HPLwt et de l’HPLdel durant cinq semaines de stockage à -80°C. De plus, l’ajout de composés chimiques tels que le KCl, le NaCl, le Na2SO4, la glycine et le glycérol permettent d’augmenter l’activité enzymatique des deux enzymes et d’améliorer les conditions de synthèse de l’hexanal et du 3Z-hexénal en diminuant la quantité d’enzyme nécessaire à leur production. / The hydroperoxide lyase (HPL) derives from a metabolic pathway named lipoxygenase pathway widely represented in plants and involved in the production of flavoring compounds (hexanal, 3Z-hexenal and 2E-hexenal). These volatile compounds are responsible for the fresh odor of cut grass known as "green note" and have a particularly interest for flavor and food industries. Their biosynthesis results from the oxygenation of linoleic and linolenic acids by lipoxygenase action to form fatty acid hydroperoxides, then of their cleavage by hydroperoxide lyase action. The processes of production currently used are highly polluting or lead to a low yield. To overcome these drawbacks, the use of recombinant enzymes in such processes constitutes an attractive alternative because they would allow producing these molecules in a more effective way, while benefiting from the "natural" label.A cDNA encoding for HPL (HPLwt) from black olive fruit was isolated, and in order to improve the enzyme solubility, the HPL deleted of its chloroplast transit peptide (HPLdel) was then produced. Both enzymes were expressed into E. coli (M15), purified by affinity chromatography, and characterized. They act exclusively on 13-hydroperoxide (13-HPL) and display an optimum pH at 7.5 and an optimum temperature at 25 °C. The bioconversion of 13-hydroperoxides of linoleic and linolenic acids in hexanal and 3Z-hexenal respectively, using HPLwt or HPLdel was studied. Conversion yields reach a maximum of 93 % and 68 % for hexanal production, and 73 % and 45 % for 3Z-hexenal when reactions were performed by HPLwt and HPLdel respectively.The enzyme stability was then studied. Conservations tests using 10 % glycerol (v/v) allows the maintenance of the entire activity of HPLwt and HPLdel during five weeks of storage at -80°C. Furthermore, the addition of chemical compounds such as KCl, NaCl, Na2SO4, glycine, and glycerol can increase the efficiency of both enzymes and improve the synthesis of hexanal and 3Z-hexenal by decreasing the amount of enzyme required to produce them.