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

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140 her work on <strong>B12</strong>-dependent propanediol degradation, with the successful identification and partial characterization one of the metabolic enzymes involved in this pathway. In the summer of 2001, she switched her focus of study to <strong>B12</strong> metabolism in humans, with specific interest in identifying the enzymes involved in AdoCbl formation, which is the basis of this dissertation. She plans to continue her academic study and intellectual development as a postdoctoral fellow.
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B12 METABOLISM IN HUMANS By NICOLE
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The work in this dissertation is de
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TABLE OF CONTENTS Page ACKNOWLEDGME
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General Molecular and Protein Metho
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LIST OF TABLES Table page 1-1. Aden
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3-7. Stoichiometry of the MSR-ATR s
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DTT dithiothreitol E. coli Escheric
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Abstract of Dissertation Presented
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CHAPTER 1 INTRODUCTION History of C
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3 (DMB). The DMB moiety is covalent
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5 and deoxyadenosine. After hydroge
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7 homocysteine recycling and methio
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methylmalonic acid. Methylmalonic a
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folate-dependent reactions includin
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tetanomorphum, P. shermanii, Euglen
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enzyme. This suggests that cob(II)a
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at the 5’-C of ATP by cob(I)alami
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iochemical studies and complementat
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1997). Both cases of the cblD disor
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23 The cblB complementation group i
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(Watkins et al. 2002). Some patient
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Chapter 2 of this dissertation repo
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Corrin Ring Dimethyl benzimidazole
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Classes Eliminases Dehydratases OH
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A) B) CH 3THF 33 MS-cob(I)alamin Me
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propanol dehydrogenase (PduQ) propa
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or from mutations that impair the a
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1989). 5-bromo-4-chloro-3-indolyl-
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41 5’-GCCGCCGGTACCGATGACGACGACAAG
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43 Growth of Adenosyltransferase Ex
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anti-rabbit IgG (γ-chain specific)
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sequence similarity to a known ATR
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ATRs were produced as fusion protei
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51 of the lacI repressor (not shown
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in which proteins bind B12, the "ba
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libraries may be particularly usefu
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57 Figure 2-1. Multiple sequence al
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Table 2-2. Specific activities of b
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61 1 2 3 4 5 25 kDa Figure 2-4. Wes
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63 (cyanocobalamin, CNCbl) and hydr
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65 5’- triphosphate (ATP), and di
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extracts of ATR 239K (160 mg protei
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Milford, MA). Samples (200 µl) wer
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Linearity of the ATR reaction 71 Th
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73 reactions was characteristic of
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75 MSR Produces little Cob(I)alamin
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Johnson et al. 2001, Saridakis et a
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AdoCbl by the MSR-ATR system. Exper
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propionyl-CoA PCC (2S)-methylmalony
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kDa 97 66 45 31 21 14 7 83 1 2 3 4
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85 Table 3-3. The MSR-ATR system se
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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
Page 119 and 120: 103 from New England Biolabs (Bever
Page 121 and 122: 105 Propionaldehyde Dehydrogenase A
Page 123 and 124: 107 fraction. Following centrifugat
Page 125 and 126: 109 antiserum obtained was specific
Page 127 and 128: 111 biochemical evidence was presen
Page 129 and 130: 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: BIOGRAPHICAL SKETCH Nicole Aurora L
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