The D282N and Y254L active-site mutants of flavocytochrome b₂ are expressed in E. coli as a mixture of holoenzyme and flavin-free protein

Kapitel in diesem Buch

1. Frontmatter I
2. PREFACE V
3. Committees VII
4. CONTENTS IX
5. THEORETICAL AND CHEMICAL APPROACH
6. Use of Molecular Orbital Calculations in Studies on Mechanisms of Enzyme Catalysis 3
7. A theoretical approach of the mechanism of C(4a)-peroxyflavin catalyzed reactions 13
8. Semiempirical calculations on properties of (iso)alloxazines in ground and excited states 23
9. Pathways for a 4^a-hydroxy-4^a,5-dihydroflavin radical in the hydroxylation of aromatics 27
10. CHEMICAL SYNTHESIS OF NEKOFLAVIN 31
11. NOVEL 5 - SUBSTITUTED 5 - DEAZAFLAVINS : SYNTHESIS AND APPLICATIONS AS ACTIVE SITE PROBES FOR FLAVOPROTEINS 35
12. D-Lactate Dehydrogenase Model. Oxidation of a-Hydroxy Acid by Functionalized Oxidation Active flavin Mimic in the Presence of Zn²⁺ and Base in t-Butanol 39
13. Mechanism of Flavin Reduction By Cu(I)-EDTA Complex 43
14. SELF-ASSOCIATION OF FLAVIN: INTERACTION OF FMN WITH ALBUMIN 47
15. BIOSYNTHESIS OF FLAVINS
16. STUDIES ON THE BIOSYNTHESIS OF FLAVINS. STRUCTURE AND MECHANISM OF 6,7-DIMETHYL-8-RIBITYLLUMAZINE SYNTHASE 53
17. BIOSYNTHESIS OF RIBOFLAVIN. CLONING, SEQUENCING, MAPPING, AND HYPEREXPRESSION OF THE GENES ribA CODING FOR GTP CYCLOHYDROLASE II AND ribC CODING FOR RIBOFLAVIN SYNTHASE OF ESCHERICHIA COLL 63
18. BIOSYNTHESIS OF RIBOFLAVIN: ENZYMATIC FORMATION OF 6,7-DIMETHYL-8-RIBITYLLUMAZINE IN Saccharomyces cerevisiae 67
19. ELECTRON MICROSCOPIC STUDIES ON THE LUMAZINE SYNTHASE/ RIBOFLAVIN SYNTHASE COMPLEX OF BACILLUS SUBTILIS 71
20. ¹⁹F NMR STUDIES ON LUMAZINE PROTEIN FROM PHOTOBACTERIUM PHOSPHOREUM 75
21. 19^F NMR STUDIES ON THE MECHANISM OF RIBOFLAVIN SYNTHASE 79
22. OXIDASES
23. STUDIES ON ACTIVE SITE MUTANTS OF SPINACH GLYCOLATE OXIDASE 85
24. HIGH-LEVEL EXPRESSION OF RAT KIDNEY HYDROXY ACID OXIDASE IN ESCHERICHIA COLI: PURIFICATION AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN 95
25. The Influence of Substrate Structure on the Redox Reactions of Monoamine Oxidases 99
26. QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIPS IN THE OXIDATION OF BENZYLAMINE ANALOGUES BY BOVINE LIVER MONOAMINE OXIDASE B 109
27. Experimental Probes of Hydrogen Tunneling in Bovine Liver Monoamine Oxidase B 117
28. Stereospecificity and Deuterium Isotope Effect in the Oxidative Deamination Catalyzed by Flavine and Non-flavine Amine Oxidases 123
29. CHARACTERIZATION OF L-ASPARTATE OXIDASE OVEREXPRESSED IN E. COLI 127
30. IDENTIFICATION OF ANOTHER SUBUNIT IN L-PHENYLALANINE OXIDASE FROM PSEUDOMONAS SP. P-501 AND THE PRIMARY STRUCTURE OF THE SUBUNIT 137
31. Kinetic Isotope Effects on Reductive Half-Reactions of Flavoprotein Oxidases 141
32. Effects of Ligands on the Reactivities of Reduced and Semiquinoid Forms of D-Amino Acid Oxidase 151
33. Thermodynamic Study of FAD Binding in D-Amino Acid Oxidase 155
34. Structure of Human D- Amino Acid Oxidase Gene 159
35. AMINO ACID SEQUENCE OF D-AMINO ACID OXIDASE FROM THE YEAST Rhodotorula gracilis 163
36. CHEMICAL MODIFICATION OF ARGEVINE GROUPS IN D-AMINO ACID OXIDASE FROM Rhodotorula gracilis Involvement in catalysis and assignment in the sequence 167
37. FUNCTIONAL AND STRUCTURAL ASPECTS OF D-AMINO ACID OXIDASE FROM Rhodotorula gracilis PROBED BY LIMITED PROTEOLYSIS 171
38. THE CRYSTAL STRUCTURE OF CHOLESTEROL OXIDASE. MECHANISTIC IMPLICATIONS FOR FAD DEPENDENT ALCOHOL OXIDATION 175
39. Nitroalkane-Oxidizing Flavoenzymes 185
40. A 340-nm Chromophore of Nitroalkane Oxidase from Fusarium oxysporum 195
41. OXYGENASES
42. LACTATE MONOOXYGENASE: STUDIES OF ACTIVE SITE MUTATIONS 201
43. The Mobile Flavin of Parahydroxybenzoate Hydroxylase - A Case for Major Structural Dynamics in Catalysis 211
44. Structures of Mutant p-Hydroxybenzoate Hydroxylases: Evidence for an Alternative Mode of Flavin Binding 221
45. Substrate and Effector Specificity of Two Active-Site Mutants of p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens 231
46. Solvent Isotope Effects on p-Hydroxybenzoate Hydroxylase 235
47. IMMUNOLOGICAL STUDIES ON THE STRUCTURE OF 4-AMINOBENZOATE HYDROXYLASE FROM AGARICUS BISPORUS 239
48. STUDIES ON p-HYDROXYPHENYLACETATE-3-HYDROXYLASE 243
49. Fluorescent Intermediates and the Reaction Mechanism of Phenol Hydroxylase 247
50. Application of pulse radiolysis to produce absorbing species of substrates for flavoprotein hydroxylases 251
51. Mammalian Flavin-Containing Monooxygenase Catalyzed Conversion of 4-Halo-NMethylanilines 255
52. 2-AMINOBENZOYL-CoA-MONOOXYGENASE/REDUCTASE, AN ENZYME WITH TWO DISTINCT FUNCTIONS AND ONE ACTIVE CENTER 259
53. Characteristic Properties and Kinetic Analysis of Neurotoxins for Porcine FAD-containing Monooxygenase 263
54. Genomic DNA Structure of a Unique Flavoprotein, 2-Nitropropane Dioxygenase from Hansenula rnrakii 267
55. DEHYDROGENASES AND ELECTRON TRANSFER
56. THREE DIMENSIONAL STRUCTURES OF ACYL-CoA DEHYDROGENASES: STRUCTURAL BASIS OF SUBSTRATE SPECIFICITY 273
57. MECHANISM OF a,ß-DEHYDROGENATION BY ACYL-COA DEHYDROGENASES 283
58. 13C- and 15N-NMR Studies of Medium-Chain Acyl-CoA Dehydrogenase from Porcine Kidney 293
59. Resonance Raman Studies on Acyl-CoA Dehydrogenases 303
60. Two New Mechanism-Based Inhibitors of the Acyl-CoA Dehydrogenases 313
61. STÜDES OF THE THERMODYNAMIC REGULATION OF MEDIUM CHAIN ACYL-CoA DEHYDROGENASE 323
62. LONG-CHAIN SPECIFIC ENZYME FROM MEDIUM-CHAIN ACYL-COA DEHYDROGENASE 327
63. Glutaryl-CoA dehydrogenase from anaerobic, benzoate degrading Pseudomonas sp.: An FAD-dependent glutaconyl-CoA decarboxylase 331
64. CRYSTALLIZATION AND PRELIMINARY X-RAY DIFFRACTION STUDY OF THE BIFUNCTIONAL FLAVOENZYME 5-HYDROXYVALERYL-COA DEHYDRATASE/DEHYDROGENASE FROM CLOSTRIDIUM AMINOVALERICUM 335
65. ESSENTIAL ARGININE RESIDUE(S) OF 3-KETOSTEROID-Δ¹- DEHYDROGENASE 339
66. Flavodoxins and Nitrogen Fixation - Structure, Electrochemistry and Post-translational Modification by Coenzyme A 343
67. REGULATION OF THE EXPRESSION OF FLAVODOXIN AND FERREDOXIN IN Anabaena PCC 7120 UNDER IRON AND NITROGEN LIMITING CONDITIONS 355
68. Crystallization and X-ray Structure Determination of E. coli Flavodoxin 359
69. Cis-trans Isomerization of the 57-58 Peptide in Crystalline Flavodoxins from C. beijerinckii 363
70. Electron-Transferring Flavoprotein from Pig Kidney Contains an AMP 367
71. The Enigma of Old Yellow Enzyme. II 371
72. STIRRING NEW INTEREST IN AN OLD ENZYME: CRYSTAL STRUCTURE OF OLD YELLOW ENZYME 381
73. EFFECT OF DELETION AND MUTATION OF OLD YELLOW ENZYME GENE FROM SACCHAROMYCES CEREVISIAE 391
74. STRUCTURE AND FUNCTION OF NADH-CYTOCHROME b5 REDUCTASE IN RELATION TO HEREDITARY METHEMOGLOBINEMIA 395
75. ROLE OF FLAVIN BINDING MOTIF, RxY(T/S), OF NADH-CYTOCHROME b₅ REDUCTASE IN ELECTRON TRANSFER REACTION 405
76. THE STRUCTURE OF HUMAN ERYTHROCYTE NADH-CYTOCHROME b₅ REDUCTASE AT 2.5Â RESOLUTION 409
77. Two Bound Forms of Ferredoxin-NADP Reductase in Chloroplast Thylakoids 413
78. CHARACTERIZATION OF ACTIVE-SITE MUTANTS OF FERREDOXIN-NADP⁺ REDUCTASE 423
79. Structural Study of Ferredoxin-NADP⁺ Reductase from Anabaena PCC 7119 and of Its Complex with NADP⁺ 431
80. Structure-Function Relationships in Ferredoxin: Site-Directed Mutagenesis and Time-Resolved Absorption Spectroscopy Applied to the Ferredoxin:Ferredoxin NADP⁺ Reductase Interaction 435
81. N-Terminal Structure of Mature Ferredoxin-NADP Reductase 439
82. Refined Crystal Structures of Native, Complexed and Reduced Forms of Spinach Ferredoxin Reductase 443
83. Overexpression of ferredoxin-NADP⁺ reductase from Anabaena sp. PCC7119 in E.coli 447
84. THE REDUCTASE ACTIVITY OP A FLAVOPROTEIN IS CHANGED INTO A SUPEROXIDE-FORMING OXIDASE BY CHEMICAL MODIFICATION 451
85. FNR -LIKE FLAVOPROTEINS OF CHEMOAUTOTROPHIC BACTERIA AND PHOTOSYNTHETIC BACTERIA 455
86. ¹H NMR investigation of NADPH-adrenoferredoxin reductase with NADP⁺ and adrenoferredoxin 459
87. One electron reduction of adrenodoxin reductase as studied by pulse radiolysis 463
88. STRUCTURE-FUNCTION STUDIES ON NADPH-CYTOCHROME P450 REDUCTASE USING UREA-PERTURBATION AND ¹⁹F NMR SPECTROSCOPY 467
89. Cerebellar Nitric Oxide Synthase Behaves as a Bi-Domain Structure 471
90. STUDIES ON NAD(P)H-QUINONE OXIDOREDUCTASE 475
91. EFFECT OF RIBOFLAVIN DEFICIENCY ON DT-DIAPHORASE 479
92. DISULFIDE REDUCTASES
93. Hybrid Molecules of Glutathione Reductase: Tools for Investigating Protein Interactions at the Dimer Interface 485
94. THE REDUCTIVE HALF REACTION OF ESCHERICHIA COLI GLUTATHIONE REDUCTASE 493
95. DETERMINATION OF THE REDOX POTENTIAL OF E. COLI GLUTATHIONE REDUCTASE USING NADH AND NAD⁺ 497
96. AN INVESTIGATION OF ENGINEERED CO-OPERATIVITY IN INTERFACE MUTANTS OF ESCHERICHIA COU GLUTATHIONE REDUCTASE 501
97. ROLE OF CONSERVED GLYCINE RESIDUES IN THE NADPH BINDING MOTIF OF GLUTATHIONE REDUCTASE 505
98. Eukaryotic Lipoamide Dehydrogenase: Molecular Genetic and Structural Aspects 509
99. Disruption of the Gene Coding for Dihydrolipoamide Dehydrogenase in Haloferax volcanii by Homologous Recombination 519
100. Redox Properties of the FAD in the Active Site Mutants C44S and C49S of Escherichia coli Lipoamide Dehydrogenase 523
101. R- AND S-DIHYDROLIPOIC ACID DERIVATIVES AS SUBSTRATES OF LIPO AMIDE DEHYDROGENASE 527
102. Comparative Study on Flavin Radical Formation of Llipoamide Dehydrogenase and Glutathione Reductase by Photoreduction 531
103. The Pyruvate Dehydrogenase Complex from Azotobacter vinelandii 535
104. The 3-dimensional structure of trypanothione reductase from Trypanosoma cruzi as a basis for drug design against Chagas' disease 545
105. MECHANISM AND STRUCTURE OF THIOREDOXIN REDUCTASE 557
106. E. COLI THIOREDOXIN REDUCTASE - POTENTIAL ACID-BASE CATALYSTS - PROPERTIES OF HIS-245 AND ASP-139 MUTANTS 567
107. RAPID REACTION KINETICS OF ESCHERICHIA COLI THIOREDOXIN REDUCTASE 571
108. 2'- Fluoro - 2'- Deoxy - Arabino - FAD: Effects On The Formation And Stability Of 2-Electron Reduced Mercuric Ion Reductase 575
109. The Alkyl Hydroperoxide Reductase of Salmonella typhimurium: Redox Activity of the Cystine Disulfide of AhpC and Evidence for Involvement in Peroxide Reduction 583
110. FLAVOPROTEIN PEROXIDE AND DISULFIDE REDUCTASES AND THEIR ROLES IN STREPTOCOCCAL OXIDATIVE METABOLISM 587
111. N⁶-(2-AMINOETHYL)-FAD: SYNTHESIS AND COENZYME ACTIVITY WITH RESPECT TO APO-NADH OXIDASE FROM THERMUS THERMOPHILICS AND THERMUS AQUATICUS 597
112. A FLAVOPROTEIN FUNCTIONAL AS NADH OXIDASE FROM Amphibacillus xylanus EpOl 601
113. COMPLEX FLAVOPROTEINS
114. Probing the Structure and Function of Flavocytochrome b² Using Protein Engineering Methods 607
115. The L-Mandelate Dehydrogenase from Rhodotorula gratninis is a Flavocytochrome b₂ 617
116. S-MANDEL ATE DEHYDROGENASE FROM PSEUDOMONAS PUTIDA: CONSTRUCTION OF A SOLUBLE CHIMERIC MUTANT OF A MEMBRANE-BOUND ENZYME 621
117. The D282N and Y254L active-site mutants of flavocytochrome b₂ are expressed in E. coli as a mixture of holoenzyme and flavin-free protein 625
118. RECONSTITUTION OF FLAVIN-FREE FLAVOCYTOCHROME b₂ WITH 5-DEAZAFMN : A CARBANION OR A HYDRIDE MECHANISM ? 629
119. Physical Studies on Phthalate Dioxygenase Reductase (PDR) 639
120. Structures of Oxidized, Reduced, and Liganded States of the Iron-Sulfur Flavoprotein, Phthalate Dioxygenase Reductase 649
121. Flavin and iron-sulfur cluster containing hydroxyacyl-CoA dehydratases (I) (R)-2-Hydroxyglutaryl-CoA dehydratases from Acidaminococcus fermentans and Fusobacterium nucleatum 659
122. Flavin and iron-sulfur containing hydroxyacyl-CoA dehydratases (II) 4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum 663
123. Glutamate synthase from AzospiriUum brasilense: structural and mechanistic studies 667
124. Cloning and Expression of Azospirillum brasilense Glutamate Synthase 675
125. Phototrophic Bacterial Flavocytochromes c 685
126. Structure and Function of the Soluble Fumarate Reductase, Flavocytochrome c, from Shewanella putrefaciens 695
127. XANTHINE DEHYDROGENASE: STRUCTURE AND PROPERTIES 699
128. THE PRIMARY STRUCTURE OF HUMAN XANTHINE DEHYDROGENASE AND CHROMOSOMAL LOCATION OF THE GENE 707
129. Overexpression and Characterization of Xanthine Dehydrogenase in a Baculovirus-Insectcell System 711
130. Redox Potentials of Milk Xanthine Dehydrogenase 715
131. Electron transfer in milk xanthine dehydrogenase as studied by pulse radiolysis 719
132. A COMPARISON OF SUBSTRATE SPECIFICITY BETWEEN MILK XANTHINE OXIDASE AND XANTHINE DEHYDROGENASE 723
133. CRYSTALLIZATION AND PRELIMINARY X-RAY DIFFRACTION OF XANTHINE OXIDASE ISOLATED FROM BOVINE MILK 727
134. NEW TIGHT BINDING INHIBITORS OF XANTHINE OXIDASE 731
135. Molecular cloning and structural characterization of the gene locus coding for mouse xanthine oxidoreductase 735
136. Mouse L929 cells are defective in the expression of xanthine oxidoreductase enzymatic activity and molybdenum (VI) salts can complement the deficit 739
137. Physicochemical Properties of Retinal Oxidase Purified from Rabbit Hepatic Cytosol 743
138. Mutagenesis of the Cysteine Residues of the FAD Domain of Nitrate Reductase 747
139. Electron Transfer and Prototropic Equilibria in Two Complex Metalloflavoproteins 751
140. THE ABSENCE OF PROTOHEME IN ACTIVE RESPIRATORY COMPLEX II OF THE THERMO ACIDOPHILIC ARCH AEON, SULFOLOBUS SP. STRAIN 7 755
141. Unique Properties of the "Rotenone Site" of NADH-Q Oxidoreductase 759
142. Reconstitution of a Cell-free Superoxide Generation of Human Neutrophil 763
143. FLAVOPROTEINS CONTAINING COVALENTLY-BOUND FLAVINS
144. Preparation and Properties of Recombinant Corynebacterial Sarcosine Oxidase 769
145. One-Step Cloning and Overexpression of the Sarcosine Oxidase Operon from Corynebacterium sp. P-1 779
146. L-GULONO-γ-LACTONE OXIDASE — cDNA CLONING AND ELUCIDATION OF THE GENETIC DEFECT IN A MUTANT RAT WITH OSTEOGENIC DISORDER 783
147. HIGH-LEVEL EXPRESSION OF RAT L-GULONO-y-LACTONE OXIDASE IN SILKWORM CELLS WITH A BACULOVIRUS VECTOR 791
148. Structures Responsible for FAD Binding and Substrate Recognition of Rat Liver Monoamine Oxidase 795
149. Vanillyl-Alcohol Oxidase from Pénicillium simplicissimum: A Novel Flavoprotein Containing 8a-(N³-histidyl)-FAD 799
150. Flavinylation of the Trimethylamine Dehydrogenase of Bacterium W₃A₁ Expressed in Escherichia coli 803
151. A New Family of Flavoenzymes ? 807
152. ENZYMES USING REDUCED FLAVIN FOR THEIR ACTIVITY
153. THE BACTERIAL LUCIFERASE REACTION: MODEL OR MAVERICK IN FLAVIN BIOCHEMISTRY? 813
154. KINETIC CONTROL OF FOLDING AND ASSEMBLY OF HETERODIMERIC BACTERIAL LUCIFERASE 823
155. KINETIC MECHANISM OF THE BACTERIAL LUCIFERASE REACTION 829
156. BACTERIAL LUCIFERASE: BIOLUMINESCENCE EMISSION USING LUMAZINES AS SUBSTRATES 839
157. Studies on Escherichia coli Chorismate Synthase 843
158. Characterization of the Vibrio harveyi NADPH:FMN Oxidoreductase Expressed in Escherichia coli 847
159. LIST OF PARTICIPANTS 851
160. AUTHOR INDEX 857
161. SUBJECT INDEX 865
162. Backmatter 891