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

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120 enzymes measured in any tissue. Similar methods could be applied for the identification of additional cDNAs involved in cobalamin metabolism. To determine whether the bovine and human cDNAs that we identified encode ATRs, the cDNAs were cloned and over expressed in E. coli. Cell extracts from the expression strains were then used for biochemical studies. Both the soluble and insoluble fractions of the crude cell extracts of bovine and human expression strains were found to have ATR activity. This suggests that the cDNAs we identified encode ATR enzymes. Additional studies were used to show that the human cDNA could function as an ATR in vivo. An S. enterica ATR mutant transformed with a plasmid-encoded source of the human ATR was used for growth studies on 1,2-propanediol supplemented with HOCbl. From these experiments, we showed that the human ATR restored wild-type phenotype to an ATR-deficient S. enterica strain proving that the human ATR can function physiologically in a heterologous host. Previous studies have shown that patients with cblB methylmalonic aciduria lack ATR activity (Fenton and Rosenberg 1981). To determine if mutations in the ATR underlie cblB methylmalonic aciduria, expression of this enzyme was monitored in normal and cblB mutant fibroblast cells. Western blot analysis using antibodies specific for the ATR showed that expression of this protein was altered in fibroblast cells from patients with cblB when compared to control cells. Additional studies conducted concurrently in another lab showed that the ATR gene (MMAB) corresponded to the cblB complementation group of methylmalonic aciduria (Dobson et al. 2002). Biochemical Characterization of the Human Adenosyltransferase In Chapter 3, we investigated the biochemical properties of the human ATR. Dobson et al. previously analyzed the MMAB gene and identified two amino acid
121 substitutions that are common polymorphic variants in control cell lines (Dobson et al. 2002). Both of these variants were independently expressed, purified, and used for biochemical analysis. In the studies conducted, the variants behaved similarly suggesting that both are normal forms of the enzyme. The kinetic properties including Vmax, and Km for ATP and cob(I)alamin were determined. The Vmax falls within the range of previously reported ATRs including CobA, PduO, and T. acidophilum ATR (Suh and Escalante- Semerena 1995, Johnson et al. 2001, Saridakis et al. 2004). The Km for cob(I)alamin (1 µM) is physiologically significant when compared to the intracellular concentration of <strong>B12</strong> in human tissue (Schneider and Stroinski 1987). The Km for ATP is low when compared to the physiological levels of this substrate but is considered relevant since ATP is required for many different reactions in human tissue. In this study, the nucleotide specificity of the human ATR variants was also analyzed. It was found that ATR variants are highly specific for ATP (the physiological substrate for adenosylation). This result is not surprising since catalysis with other nucleotide substrates (GTP, CTP, and UTP) resulting in GCbl, CCbl, and UCbl are generally inhibitory to AdoCbl-dependent enzymes and would be counter-productive (Toraya and Mori 1999). Establishing the biochemical properties of control ATRs provides a basis of comparison for the characterization of disease causing variants of this enzyme. Methionine Synthase Reductase is a Cob(II)alamin Reductase for the Human Adenosyltransferase In addition to analyzing the biochemical properties of the human ATR, we also investigated whether human MSR can act as a cob(II)alamin reductase for the synthesis of AdoCbl by the ATR (Chapter 3). Previous studies have shown that MSR functions as
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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
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: CHAPTER 5 CONCLUSIONS In humans, co
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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