B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

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2 IDENTIFICATION OF THE HUMAN AND BOVINE ATP:COB(I)ALAMIN ADENOSYLTRANSFERASE CDNAS BASED ON COMPLEMENTATION OF A BACTERIAL MUTANT........................................................................................36 Introduction.................................................................................................................36 Materials and Methods ...............................................................................................38 Chemicals and Reagents......................................................................................38 Bacterial Strains and Growth Media ...................................................................39 General Protein and Molecular Methods and ATR Assays.................................39 P22 Transductions ...............................................................................................39 Screening the Bovine and Human Liver cDNA Libraries...................................40 Cloning the Bovine Adenosyltransferase Coding Sequence for High-Level Expression...............................................................................40 Cloning the Human Adenosyltransferase Coding Sequence for High-Level Expression...............................................................................41 Cloning the Human Adenosyltransferase Coding Sequence for Complementation Studies ..........................................................................42 Growth of Adenosyltransferase Expression Strains and Preparation of Cell Extracts ................................................................................................43 Growth Curves.....................................................................................................43 Western Blots ......................................................................................................44 DNA Sequencing and Analysis...........................................................................45 Results.........................................................................................................................45 Screening of the Bovine and Human Liver cDNA Libraries for Clones that Express Adenosyltransferase Activity .............................................................45 Identification of a Human Gene Related to the Bovine Adenosyltransferase cDNA ...............................................................................................................47 Putative Subcellular Localization of Adenosyltransferase Enzymes ..................48 High-Level Expression of the Bovine and Human Adenosyltransferases in E. coli...........................................................................................................48 Assay of the Bovine and Human Adenosyltransferase Fusion Proteins for ATR Activity..............................................................................................49 Complementation of an S. enterica Mutant Deficient in ATR Activity by a Human Adenosyltransferase cDNA Clone ......................................................50 Level of Human Adenosyltransferase Enzyme in Normal and cblB Mutant Human Skin Fibroblasts...................................................................................51 Conserved Amino Acids and Distribution of Adenosyltransferase Enzymes.....52 Discussion...................................................................................................................53 3 PURIFICATION AND INITIAL CHARACTERIZATION OF THE HUMAN ATP:COB(I)ALAMIN ADENOSYLTRANSFERASE AND ITS INTERACTION WITH METHIONINE SYNTHASE REDUCTASE .................................................62 Introduction.................................................................................................................62 Materials and Methods ...............................................................................................64 Chemicals and Reagents......................................................................................64 Bacterial Strains and Growth Media ...................................................................65 vi
General Molecular and Protein Methods.............................................................65 ATP:cob(I)alamin Adenosyltransferase Assays..................................................65 Construction of ATR Expression Strains ............................................................66 Purification of the Human ATR Variants............................................................66 MSR-ATR Assay.................................................................................................68 Measurement of Cob(I)alamin with Iodoacetate.................................................68 Separation and Quantification of Cobalamins by HPLC ....................................68 Effect of Ionic Strength on the MSR-ATR System.............................................69 DNA Sequencing.................................................................................................69 Results.........................................................................................................................70 Purification of the Human ATR Variants............................................................70 Linearity of the ATR reaction .............................................................................71 ATR Reaction Requirements...............................................................................71 Alternative Nucleotide Donors............................................................................71 Km and Vmax Values for ATR 239K and 239M ...................................................71 MSR Reduces Cob(II)alamin to Cob(I)alamin for AdoCbl Synthesis................72 Cob(I)alamin is Sequestered by the MSR-ATR System .....................................73 MSR Produces little Cob(I)alamin in the Absence of the ATR ..........................75 Stoichiometry of the MSR-ATR system .............................................................75 Ionic Strength Dependence of Cobalamin Reduction and Adenosylation by the MSR-ATR System ................................................................................75 Discussion...................................................................................................................76 4 PDUP IS A COENZYME-A-ACYLATING PROPIONALDEHYDE DEHYDROGENASE ASSOCIATED WITH THE POLYHEDRAL BODIES INVOLVED IN B12-DEPENDENT 1,2-PROPANEDIOL DEGRADATION BY SALMONELLA ENTERICA SEROVAR TYPHIMURIUM LT2...............................92 Introduction.................................................................................................................92 Materials and Methods ...............................................................................................95 Bacterial Strains, Media, and Growth Conditions...............................................95 General Molecular Methods ................................................................................95 General Protein Methods.....................................................................................95 P22 Transduction.................................................................................................96 Cloning pduP into pLAC22.................................................................................96 Construction of His8-PduP ..................................................................................97 Purification of His8-PduP under Denaturing and Nondenaturing Conditions.....97 Propionaldehyde Dehydrogenase Assays............................................................98 Identification of Propionyl-CoA a Product of the Propionaldehyde Dehydrogenase Reaction .................................................................................99 Construction of a Nonpolar pduP Deletion.......................................................100 Antibody Preparation.........................................................................................101 Western Blots ....................................................................................................101 Electron Microscopy .........................................................................................102 DNA Sequencing and Analysis.........................................................................102 Chemicals and Reagents....................................................................................102 Results.......................................................................................................................103 vii
Page 1 and 2: B12 METABOLISM IN HUMANS By NICOLE
Page 3 and 4: The work in this dissertation is de
Page 5: TABLE OF CONTENTS Page ACKNOWLEDGME
Page 9 and 10: LIST OF TABLES Table page 1-1. Aden
Page 11 and 12: 3-7. Stoichiometry of the MSR-ATR s
Page 13 and 14: DTT dithiothreitol E. coli Escheric
Page 15 and 16: Abstract of Dissertation Presented
Page 17 and 18: CHAPTER 1 INTRODUCTION History of C
Page 19 and 20: 3 (DMB). The DMB moiety is covalent
Page 21 and 22: 5 and deoxyadenosine. After hydroge
Page 23 and 24: 7 homocysteine recycling and methio
Page 25 and 26: methylmalonic acid. Methylmalonic a
Page 27 and 28: folate-dependent reactions includin
Page 29 and 30: tetanomorphum, P. shermanii, Euglen
Page 31 and 32: enzyme. This suggests that cob(II)a
Page 33 and 34: at the 5’-C of ATP by cob(I)alami
Page 35 and 36: iochemical studies and complementat
Page 37 and 38: 1997). Both cases of the cblD disor
Page 39 and 40: 23 The cblB complementation group i
Page 41 and 42: (Watkins et al. 2002). Some patient
Page 43 and 44: Chapter 2 of this dissertation repo
Page 45 and 46: Corrin Ring Dimethyl benzimidazole
Page 47 and 48: Classes Eliminases Dehydratases OH
Page 49 and 50: A) B) CH 3THF 33 MS-cob(I)alamin Me
Page 51 and 52: propanol dehydrogenase (PduQ) propa
Page 53 and 54: or from mutations that impair the a
Page 55 and 56: 1989). 5-bromo-4-chloro-3-indolyl-
Page 57 and 58:
41 5’-GCCGCCGGTACCGATGACGACGACAAG
Page 59 and 60:
43 Growth of Adenosyltransferase Ex
Page 61 and 62:
anti-rabbit IgG (γ-chain specific)
Page 63 and 64:
sequence similarity to a known ATR
Page 65 and 66:
ATRs were produced as fusion protei
Page 67 and 68:
51 of the lacI repressor (not shown
Page 69 and 70:
in which proteins bind B12, the "ba
Page 71 and 72:
libraries may be particularly usefu
Page 73 and 74:
57 Figure 2-1. Multiple sequence al
Page 75 and 76:
Table 2-2. Specific activities of b
Page 77 and 78:
61 1 2 3 4 5 25 kDa Figure 2-4. Wes
Page 79 and 80:
63 (cyanocobalamin, CNCbl) and hydr
Page 81 and 82:
65 5’- triphosphate (ATP), and di
Page 83 and 84:
extracts of ATR 239K (160 mg protei
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Milford, MA). Samples (200 µl) wer
Page 87 and 88:
Linearity of the ATR reaction 71 Th
Page 89 and 90:
73 reactions was characteristic of
Page 91 and 92:
75 MSR Produces little Cob(I)alamin
Page 93 and 94:
Johnson et al. 2001, Saridakis et a
Page 95 and 96:
AdoCbl by the MSR-ATR system. Exper
Page 97 and 98:
propionyl-CoA PCC (2S)-methylmalony
Page 99 and 100:
kDa 97 66 45 31 21 14 7 83 1 2 3 4
Page 101 and 102:
85 Table 3-3. The MSR-ATR system se
Page 103 and 104:
Absorbance 0.5 0.4 0.3 0.2 0.1 0.0
Page 105 and 106:
Wavelength, (525 nm) 0.24 0.23 0.22
Page 107 and 108:
Activity, (nmol min-1 Activity, (nm
Page 109 and 110:
al. 1988). The aldehyde is then dis
Page 111 and 112:
CoA-acylating propionaldehyde dehyd
Page 113 and 114:
97 previously published DNA sequenc
Page 115 and 116:
mM CHES (2-[N-cyclohexylamino]ethan
Page 117 and 118:
101 For the conjugation step, strai
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103 from New England Biolabs (Bever
Page 121 and 122:
105 Propionaldehyde Dehydrogenase A
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107 fraction. Following centrifugat
Page 125 and 126:
109 antiserum obtained was specific
Page 127 and 128:
111 biochemical evidence was presen
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113 Table 4-1. Bacterial strains Sp
Page 131 and 132:
115 Table 4-3. Propionaldehyde dehy
Page 133 and 134:
kDa 209 80 49 35 29 117 1 2 3 4 Fig
Page 135 and 136:
CHAPTER 5 CONCLUSIONS In humans, co
Page 137 and 138:
121 substitutions that are common p
Page 139 and 140:
123 Studies also indicated that hum
Page 141 and 142:
LIST OF REFERENCES Aberg, A., S. Ha
Page 143 and 144:
127 Bradford, M. 1976. A rapid and
Page 145 and 146:
129 Fenton, W. A. and L. E. Rosenbe
Page 147 and 148:
131 Johnson, C. L. V. J., E. Pechon
Page 149 and 150:
133 Mellman, I., H. F. Willard and
Page 151 and 152:
135 Rossi, M., J. P. Glusker, L. Ra
Page 153 and 154:
137 Vlasie, M., S. Chowdhury and R.
Page 155 and 156:
BIOGRAPHICAL SKETCH Nicole Aurora L
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