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

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122 a cob(II)alamin reductase for reductive activation of MS (Olteanu and Banerjee 2001). Here, an MSR-ATR linked assay was developed and this provided biochemical evidence that MSR can also function as a cob(II)alamin reductase for AdoCbl synthesis. The rate of AdoCbl formation in these assays (0.96 nmol min -1 ) is physiologically significant since humans require only small amounts of cobalamin (Schneider 1987). This rate is also significant when compared to previous measurements of AdoCbl formed in human fibroblast cells (0.021 pmol min -1 mg -1 ) (Fenton and Rosenberg 1981). In mammalian systems, MSR is a cytosolic protein while ATR is localized to the mitochondria. In these studies we demonstrated that MSR is as a cob(II)alamin reductase for the ATR in vitro. The proposed requirement of MSR for AdoCbl synthesis necessitates the presence of MSR in both the cytoplasm and the mitochondria. Recent studies have shown that alternative transcript splicing of MSR produces protein with and without a mitochondrial targeting sequence (Leclerc et al. 1999). These findings strengthen the hypothesis that MSR has dual functionality in the cytoplasm and the mitochondria. Investigations also suggest an interaction between MSR and ATR. In MSR-ATR linked assays containing iodoacetate (an alkylating agent that forms CMCbl in the presence of cob(I)alamin), very little CMCbl was formed suggesting that MSR does not release cob(I)alamin free in solution. In addition, HPLC analyses showed that AdoCbl formation was favored even when a large excess of iodoacetate was added to the assays. These studies indicate that an interaction between MSR and ATR triggers cobalamin reduction.
123 Studies also indicated that humans have redundant systems for mitochondrial cob(II)alamin reduction. Prior complementation analysis demonstrated that cblE (MSR) disorders usually result in homocystinuria without methylmalonic aciduria with only one reported case having both homocystinuria and mild methylmalonic aciduria (Wilson et al. 1999). The fact that most cblE patients lack methylmalonic aciduria suggests that there are redundant mitochondrial cob(II)alamin reductase(s) involved in AdoCbl synthesis. There is precedence for redundant cobalamin reductases in bacteria. The role of MSR could be to provide extra capacity under certain conditions, such as a high protein diet, rich in amino acids that are metabolized via the propionyl-CoA pathway. Future Experimentation The development of the screening method employed in this study can be used in the future as a general technique for the isolation of other cDNAs involved in cobalamin metabolism. Salmonella is an excellent model system for these studies since AdoCbl-dependent diol dehydratase for 1,2-propanediol degradation has been well studied. Once other enzymes involved in cobalamin metabolism are identified in S. enterica, strains deficient in these activities can be used to screen mammalian expression libraries by complementation. Hence, this technique may allow for the identification of other genes crucial for cobalamin metabolism in eukaryotic systems. Now that a purification method has been developed for the ATR, efforts can be focused on crystallizing this protein. Determining the three dimensional structure of the ATR will allow us to understand how mutations in this enzyme lead to dysfunction by comparing amino acid residues in normal and mutant ATRs and mapping the altered amino acid residues onto the structure. Potentially, this process would help lend insight in identifying the residues of the ATR that are crucial for enzymatic activity.
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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
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: 121 substitutions that are common p
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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