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

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educed cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for the MSR enzyme. 28 Chapter 4 of this dissertation is not directly related to <strong>B12</strong> metabolism in humans, but was the research performed during my first two years in Dr. Bobik’s Laboratory. This work focused on the identification of PduP, a coenzyme-A-acylating propionaldehyde dehydrogenase associated with polyhedral bodies involved in AdoCbl-dependent 1,2-propanediol degradation by S. enterica. A PCR based method was used to construct a precise nonpolar chromosomal deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal media and expressed 105-fold less propionaldehyde dehydrogenase activity than did wild-type S. enterica grown under similar conditions. An E. coli strain was constructed for high-level production of His8-PduP, which was purified by nickel-affinity chromatography and was shown to have propionaldehyde dehydrogenase activity. Reverse-phase High Performance Liquid Chromatography (HPLC) and mass spectrometry were used to verify the product of the PduP reaction. Immunogold electron microscopy was used to show that PduP is associated with a polyhedral organelle involved in AdoCbl-dependent 1,2-propanediol degradation.
Corrin Ring Dimethyl benzimidazole H H2NC 2NC O H O 3C H3C O H H2NC 2NC Aminopropanol H H3C 3C H H3C 3C H H3C 3C C C H H3C 3C H H2NC 2NC CH C C N N Co 3+ Co 3+ C C N N O HN A O C D C C C C C C C H H3C 3C H C CH 3 R C CH 3 N N OH 29 R= upper β-ligand H H3C 3C CNH 2 C C CNH 2 O O O B O O C C P O O CH CH CH2OH 2OH CH 3 CH 3 O CNH 2 R= Active Adenosyl AdoCbl Methyl CH CH3Cbl 3Cbl R= Inactive Cyano CNCbl, vitamin B <strong>B12</strong> 12 Hydroxy HOCbl Glutathionyl GSCbl N NH 2 N OH CH 2 R R O N N OH R CH 3 C N R OH R GS Figure 1-1. Structure of cobalamins. The major parts of cobalamin are labeled: upper β-ligand, corrin ring, aminopropanol, and dimethylbenzimidazole. The four pyrrole rings of the corrin are labeled with capital letters. The upper ligand can be any of the groups listed in the legend. Active and inactive forms of cobalamin are dependent on the upper ligand.
Page 1 and 2: B12 METABOLISM IN HUMANS By NICOLE
Page 3 and 4: The work in this dissertation is de
Page 5 and 6: TABLE OF CONTENTS Page ACKNOWLEDGME
Page 7 and 8: General Molecular and Protein Metho
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: Chapter 2 of this dissertation repo
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
Page 85 and 86: 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
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 and 156:
BIOGRAPHICAL SKETCH Nicole Aurora L
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