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University Microfilms INFORMATION TO USERS This dissertation was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity. 2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. Wher, a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again - beginning below the first row and continuing on until complete. 4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced. University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company 73-9148 CONRAD, Robert Stanley, 1941- CATABOLISM OF ISOLEUCINE BY PSEUDOMONAS PUTIDA. The University of OklahcifiJ, Ph.D'., 1972 Microbiology University Microfilms, A XERCKCompany, Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. THE UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE CATABOLISM OF ISOLEUCINE BY PSEUDOMONAS PUTIDA A DISSERTATION SUBMITTED TO THE GRADUATE FACULH in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY BY ROBERT STANLEY CONRAD Oklahoma City, Oklahoma 1972 CATABOLISM OF ISOLEUCINE BY PSEUDOMONAS PUTIDA APPROVED BY L S J M . / / \ i C c I t- - d i s s e r t Xt i o n ^committee PLEASE NOTE: Some pages may have indistinct print. FiImed as received. University Microfilms, A Xerox E d u c a tio n Company ACKNOWLEDGMENTS The author wishes to express his gratitude to J. R. Sokatch, Ph.D., for his support and professional guidance. The encouragement and assistance given the author by faculty members of the Department of Microbiology and Immunology are gratefully acknowledged. iii TABLE OF CONTENTS Page LIST OF TABLES........................................... v LIST OF ILLUSTRATIONS...................................... vl LIST OF ABBREVIATIONS..................................... vii Chapter I. INTRODUCTION...................................... 1 II. MATERIALS AND METHODS ............................ 9 III. R E S U L T S ........................................... 23 IV. DISCUSSION......................................... 52 V. S U M M A R Y ........................................... 56 LITERATURE CITED ......................................... 58 IV LIST OF TABLES Table Page 1. Growth Rates of 2» putida on Branched-Chain Amino Acids. 24 2. Relief of L-Leucine Toxicity .......................... 25 3. Cell Yields of 2» Putida Grown on Different Substrates . 26 4. Induction of Catabolic Enzymes in 2- Putida............... 30 3. Purification of 2-Methyl-3-Hydroxybutyryl-CoA Dehydrogenase of 2* Putida ......................... 31 6. Substrate Specificity of Purified 2-Methyl-3- Hydroxybutyryl-CoA Dehydrogenase .................... 37 7. Thin Layer Chromatography of 2 ,4-Dinitrophenylhydrazones of 2-Methyl-3-Hydroxybutyryl-CoA Dehydrogenase End P r o d u c t ........................................... 39 8. Induction of 2-Methyl-3-Hydroxybutyryl-CoA Dehydrogenase and Tiglyl-CoA Hydrase in 2- Putida.................... 41 9. Induction of Tiglyl-CoA Hydrase in 2* putida ........... 42 10. Purification of Tiglyl-CoA Hydrase of 2* Putida........... 43 11. Induction of Cleavage Enzymes in 2* P u t i d a ............... 45 12. Acetyl-CoA Formation from 2-Methylacetoacetyl-CoA..........46 13. Induction of Isoleucine Catabolic Enzymes in 2* putida . 48 14. Comparison of Isoleucine Catabolic Enzymes in 2* putida Grown on Different Carbon Sources...................... 49 15. Comparison of Inducible Isoleucine Catabolic Enzymes in 2" putida Grown on Proposed Intermediates ......... 50 LIST OF ILLUSTRATIONS Figure Page 1. Proposed Pathway for Isoleucine Catabolism in Pseudomonas putida................................ 3 2. The Pathway of Valine Catabolism in Pseudomonas ........ 5 3. Proposed Pathway for Leucine Catabolism in Pseudomonas putida................................ 6 4. Schematic Representation of 2-Methylacetoacetyl-CoA Cleavage Enzyme Assay based on the reduction of NAD . 18 5. Protein Profile of the Eluate from DEAE-Cellulose Column. Proteins were insoluble in 30-50% ammonium sulfate........................................... 32 6. Separation of 2-Methyl-3-Hydroxybutyryl-CoA Dehydro­ genase and Tiglyl-CoA Hydrase by Isoelectric Focusing with pH Gradient from 3-10........................... 33 7. Electropherograms of Fractions Containing 2-Methyl-3- Hydroxybutyryl-CoA Dehydrogenase Activity. Fractions were as follows: (A) DEAE-cellulose column chromato­ graphy, (B) Isoelectric focusing, (C) Sephadex G-150, and (D) Control.................................... 34 8. Electropherograms of Partially Purified Isoleucine Catabolic Enzymes. (A) 2-Methyl-3-hydroxybutyryl- CoA dehydrogenase, (B) Tiglyl-CoA hydrase, (C) 2- Methyl-3-hydroxybutyryl-CoA dehydrogenase plus tiglyl-CoA hydrase, and (D) Control ................ 35 9. Catabolism of Isoleucine, Valine, and Leucine .......... 54 vi LIST OF ABBREVIATIONS ATP - adenosine triphosphate C - centigrade CoA, CoASH - coenzyme A 2,6-DCPIP - 2,6-dichlorophenolindophenol 2,4-DNP - 2,4-dinitrophenylhydrazine g - gram or gravity L - liter yg - microgram mg - milligram ml - milliliter mM - millimolar n - nano NAD - nicotinamide adenine dinucleotide (oxidized) NADH - nicotinamide adenine dinucleotide (reduced) Tris - tris(hydroxymethyl)aminomethane vii THE CATABOLISM OF ISOLEUCINE IN PSEUDOMONAS PUTIDA CHAPTER I INTRODUCTION Background The inability to catabolize branched-chain amino acids be­ came a matter of clinical concern after Menkes et showed the rela­ tionship between this deficiency and progressive familial infantile cerebral dysfunction (23). The so-called "Maple Syrup Urine Disease" that he described is a devastating disease in children. The normal prognosis includes intense multiple neurological manifestations and early death. The characteristic maple syrup odor present in the urine of afflicted children is due to the increased excretion of branched- chain amino acids and their corresponding keto acids. Connelly et al. demonstrated that bovine liver tissues normally oxidatively decarboxy- lated the keto acids of isoleucine, and leucine by means of a-keto- isocaproic; a-keto-B-methylvaleric acid dehydrogenase (3, 5). The keto acid of valine, 2-ketoisovalerate, was metabolized by a separate enzyme. A block in these enzymes could therefore produce elevated cellular levels of branched-chain amino acids and their keto acid ana­ logues. Schulman et al. (32) and Goedde_et al. (11) reported that cultured leukocytes and fibroblasts from maple syrup urine diseased 1 2 patients had a deficiency in branched-chain keto acid dehydrogenase activity that was consistent with clinical manifestations. Goedde et al. measured enzyme activity by incubating labelled ketoacids with enzyme. The labelled CO^ liberated was absorbed on filter paper sat­ urated with KOH and then quantitatively estimated in a liquid scin­ tillation spectrometer. He postulated that children completely lacking decarboxylase activity were homozygous for the mutated allele of the autosomal gene which normally controls the synthesis of branched chain keto acid dehydrogenase. According to his hypothesis heterozygotes would have an intermediate decarboxylase capability and corresponding mild form of disease expression. Other branched-chain amino acid metabolites have been impli­ cated in related metabolic disorders. Stokke et al. described one patient deficient in methylcrotonyl-CoA carboxylase activity (a leu­ cine catabolic enzyme) (38). Daum et al. found 2-methyl-3-hydroxy- butyrate and 2-methylacetoacetate in the urine of one patient suffering from a mild form of maple syrup urine disease (8). Since both of these metabolites are proposed intermediates of isoleucine catabolism past the branched-chain keto acid dehydrogenase step, it is quite likely that genetic lesions in catabolic enzymes specific for isoleucine are responsible for metabolic diseases. The proposed enzymatic reactions by which putida cata- bolizes isoleucine to acetyl-CoA and propionyl-CoA is shown in Figure 1. Some of these reactions have been previously demonstrated in Pseudomonas. Norton and Sokatch found that L-isoleucine and 2-keto- glutarate were transaminated by branched-chain amino
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