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Title: Studies of enzymes involved in mevalonate pathway and sugar modification
Other Titles: Can yu jia qiang wu suan tu jing yu tang xiu shi mei de yan jiu
Authors: Chu, Xiusheng (楚秀生)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2007
Publisher: City University of Hong Kong
Subjects: Aminoglycosides
Mevalonic acid
Notes: CityU Call Number: QP601.C48 2007
Includes bibliographical references (leaves 259-295)
Thesis (Ph.D.)--City University of Hong Kong, 2007
xxxii, 295 leaves : ill. (some col.) ; 30 cm.
Type: Thesis
Abstract: Isoprenoids existing naturally and universally in organisms consist of lots of important metabolic products such as steroids, phytosterols, cholorophylls, cholesterol, tocopherols, ubiquinone, heme A, dolichol, vitamin K2 and so on. Most of them are very important substances in the regulation of the development and growth of organisms. So far, more than 30,000 naturally occurring terpenes and terpenoids have been described. All isoprenoids are biosynthesized through an important biosynthetic pathway called the mevalonate pathway. In microorganisms and plants, the biosynthesis of isoprenoid derivatives such as terpenes and terpenoids is controlled by a series of reactions catalyzed by enzymes including mevalonate kinase, phosphomevalonate kinase, mevalonate-5-diphosphate decarboxylase, farnesyl pyrophosphate synthase and other enzymes; in animal cells, these enzymes are involved in the biosynthesis of cholesterol and steroid hormones. In human being, several diseases are related to this pathway, such as hyperimmunoglobulinemia D/periodic fever syndrome (HIDS) and cardiovascular disease. The mevalonate pathway has been the target for the treatment of human cardiovascular disease. It has reported recently that it is also an important target for the development of new drugs to control drug-resistant bacteria and anti-cancer therapy. Galactokinase, involved in the conversion of -D-galactose to glucose-1-phosphate, belongs to the same GHMP kinase superfamily as mevalonate kinase, phosphomevalonate kinase and mevalonate-5-diphosphate decarboxylase. Galactokinase has attracted significant research attention for more than 45 years partially because of its important metabolic role and the fact that defects in the human enzyme can result in galactosemia. The absence of functional galactokinase results in the buildup of unmetabolized galactose in cells and in blood plasma. The human galactosemia type II disease, the main symptom of which is early onset cataracts, is resulted from approximately 20 mutations of human galactokinase. It has been reported that the build up of the toxic galactose-1-phosphate can not only cause cataracts, but also result in damage to the brain, liver and kidneys. This effect cannot be reversed or even completely prevented by the exclusion of galactose and lactose from the diet. Therefore, it is urgent to find out effective inhibitor of galactokinase for curing the disease. Aminoglycosides like galactose belong to sugar substances. They are one of the major classes of antibiotics. The emergence of bacteria resistantance to aminoglycoside antibiotics has become a major problem over recent years, although acquired resistance has existed since the advent of antibiotics. Bacteria infection has claimed over 20 million people’s life worldwide by the end of last century mainly because of antibiotic resistance. The most prevalent source of clinically relevant resistance is conferred by the aminoglycoside structure modification by aminoglycoside-modifying enzymes. These enzymes are broadly classified as N-acetyltransferases (AACs) that transfer an acetyl group from acetyl-CoA, O-adenyltransferases (ANTs) that transfer an adenyl group from ATP, and O-phosphotransferases (APHs) that transfer a phosphate group from ATP, to aminoglycosides respectively, and they comprise over 50 different enzymes. Many compounds or analogs of aminoglycoside-modifying enzymes with the efficacy from excellent to poor substrate have been designed and synthesized. However, there are no clinical successful stories for inhibition of aminoglycoside resistance enzymes so far. The difficulty arises from the presence of so many different enzymes to be inhibited and also from the fact that the biochemical mechanism of many of these enzymes has not been studied in any meaningful detail. In this thesis, we mainly reported our studies of several enzymes including mevalonate kinase, phosphomevalonate kinase, mevalonate-5-diphosphate decarboxylase, farnesyl pyrophosphate synthase in the mevalonate pathway, and galactokinase, aminoglycoside N-acetyltransferase AAC(6')-Ii, aminoglycoside O-phosphotransferase APH(3')-IIIa, bifunctional AAC(6')APH(2"), and truncated APH(2")-Ia for sugar-modification. Through characterization of enzymes and their interactions with substrate analogues, we hope to find out effective inhibitors targeting corresponding enzymes, useful information for design of new inhibitors and effective screening or detecting methods for substrate analogues. In the project about enzymes in the mevalonate pathway, we subcloned M. jannaschii mevalonate kinase gene, and constructed six mutants including C107A, C107S, C281A, C281S, C107A/C281A and C107S/C281S related to a disulfide bond that is proposed to contribute to the thermostability and conformation stability of M. jannaschii mevalonate kinase. A series of experiments were carried out focusing on the contribution provided by a disulfide bond. Results show that the C107-C281 disulfide bridge is one of the key elements for maintaining the thermostability, the integrity of ATP-binding site, resistance to trypsin digestion and urea denaturation of M. jannaschii mevalonate kinase. We cloned rat mevalonate kinase gene and farnesyl pyrophosphate synthase gene from a rat cDNA library, and constructed ten mevalonate kinase mutants including N55A, N55S, N104A, N104D, N104S, S108A, S108N, S135A, S135N and N55A/S108N related to an ATP binding site. We also constructed mutant A141C of mevalonate kinase that is proposed to form an engineered disulfide bond contributing to the thermostability and structure stability of rat mevalonate kinase. Our results obtained from analysis of kinetic study and TNP-ATP fluorescence study indicate that four amino acids N55, N104, S108 and S135 are necessary to the ATP-binding site of mevalonate kinase, and that mutant A141C has a new disulfide bridge that has enhanced thermostability, moderate resistance to trypsin digestion and urea denaturation. From Streptococcus pneumoniae genomic DNA, we cloned four genes including mevalonate kinase (StpMVK) gene, phosphomevalonate kinase (StpPMK) gene, mevalonate-5-diphosphate decarboxylase (StpMDD) gene and farnesyl pyrophosphate synthase (StpFPPS) gene. Kinetic and inhibition studies with substrate analogues were carried out. We found that four substrate analogues including 2-fluoromevalonate-5-diphosphate, 2,2-difluoromevalonate-5-diphosphate, 3-ethyl-3,5-dihydroxy-pentanate-5-diphosphate, and P’-geranyl-2-fluoromevalonate-5-diphosphate are relatively effective inhibitors of StpMDD. The substrate analogue P’-geranyl 3,5-dihydroxy-3-methyl-pentanate-5-diphosphate is a relatively effective trifunctional inhibitor targeting StpMVK, StpPMK and StpMDD. We also found that quercetin is more effective inhibitor than genistein, and StpMDD is more easily inhibited by quercetin than other ATP-dependent enzymes in mevalonate pathway. Therefore, these relatively effective inhibitors as well as quercetin may be used for treating pneumonia caused by infection of S. pneumoniae. In the project about the enzymes involved in sugar modifications, we cloned rat galactokinase gene from rat cDNA library, and constructed six mutants including R37A, Y236A, Y236F, D186A, D186E and D186N involved in interactions with galactose at galactose-binding site, and five mutants including W106A, Y109A, Y109F, Y109H and Y109W involved in interactions with ATP at the ATP-binding site. Kinetic and inhibition studies with lots of substrate analogues were carried out, and the ATP-binding site was further investigated through TNP-ATP titration. In the present study, we found that rat galactokinase can catalyze -D-fucose, -L-arabinose, -D-talose, 3-deoxy-D-galactose and 1-amino-1-deoxy--D-galactose. Results of mutation studies showed that the noncovalent stacking interaction of the indole ring of W106 with adenine base is not an excellent interaction force in the stabilization of ATP, while D186 is absolutely necessary in the catalysis of galactose. Analysis of kinetic data showed that the configuration of the hydroxyl group at C4 and C3 is very important to the substrate-binding capacity, and that rat galactokinase can still catalyze the substrate analogue with the removal or the changed configuration of a hydroxyl group or substitution by small functional group at C2. Inhibition studies showed that fluoro -D-galactopyranoside is relatively effective inhibitor of rat galactokinase. However, quercetin with relatively high affinity to rat galactokinase, or even other known, or unknown flavanoids isolated from Chinese medicinal herbs, may become useful inhibitor, or a potential lead for the development of new useful compounds, to treat patients with galactosemia. In the present study, we also subcloned APH(3')-IIIa gene, bifunctional enzyme AAC(6')APH(2") gene, truncated APH(2")-Ia gene, and constructed APH(3')-IIIa mutants D126E and D126N, and AAC(6')-Ii mutants S121C and S135C. Kinetic studies and a series of experiments for the analysis of substrate analogue through the study of substrate-enzyme binding were carried out. A relatively effective method for substrate screening or detection was developed with a mutant AAC(6')-Ii S121C labeled by fluorescein-5-maleimide, which can not only be used for substrate screening, but also may be further developed to a biosensor for detection of aminoglycoside antibiotic present in milk. In conclusion, this thesis mainly describes the functional studies of twelve enzymes involved in mevalonate pathway and sugar modifications. The studies of their interactions with substrates and substrate analogues, and mutation studies of enzymes increased our understanding of the importance of enzyme integrity for their catalytic reactions. The introduction of a disulfide bond into rat mevalonate kinase was obtained through site-directed mutagenesis. Five relatively effective inhibitors as well as quercetin were found to inactivate S. pneumoniae enzymes in mevalonate pathway, which may be used for treating pneumonia caused by infection of this organism. Kinetic analysis of rat galactokinase provides useful information for the design or development of effective inhibitors. It was showed that quercetin might be a useful reagent to treat patients with galactosemia. In addition, a relatively effective method for screen and detection of aminoglycoside antibiotics was developed.
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