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

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78 cob(II)alamin to AdoCbl at a physiologically relevant rate. To our knowledge, this is the first reported evidence that MSR can catalyze the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis. In addition to its role in AdoCbl synthesis, prior studies showed that MSR also reduces MS-bound cob(II)alamin to cob(I)alamin for the reductive activation of MS (Leclerc et al. 1998, Olteanu and Banerjee 2001). It is known that MS activation occurs in the cytoplasm but that AdoCbl synthesis occurs in the mitochondrion. Therefore, to carry this dual function MSR must reside in both compartments. Interestingly, recent studies have indicated that alternative transcript splicing leads to the production of MSR enzymes with and without mitochondrial targeting sequences providing further evidence of a dual role of this enzyme (Leclerc et al. 1999). Findings reported here which indicate dual functionality for MSR bring to light an interesting parallel between cobalamin reduction in bacteria and in humans. Biochemical evidence indicated that the E. coli flavodoxin (FldA) catalyzes the reduction of cob(II)alamin to cob(I)alamin for both the reductive activation of MS (MetH) and the synthesis of AdoCbl by the bacterial ATR (CobA) (Fujii and Huennekens 1974, Fujii et al. 1977, Fonseca et al. 2002). Similarly, human MSR reduces cob(II)alamin for both MS activation (Leclerc et al. 1998, Olteanu and Banerjee 2001) and AdoCbl synthesis by the human ATR (this study). Thus, although the human MSR and ATR lack significant sequence similarity to their bacterial counterparts (FldA and CobA), evolution appears to have driven analogous dual physiological roles for both FldA and MSR enzymes. In this report, we also conducted studies to determine whether cob(I)alamin was sequestered or released “free” in solution during the conversion of cob(II)alamin to
AdoCbl by the MSR-ATR system. Experiments in which iodoacetate was used as a chemical trap indicated that cob(I)alamin was sequestered. This is likely to be 79 physiologically important. Cob(I)alamin is one of the strongest nucleophiles that exists in aqueous solution and an extremely strong reductant (E°' = -0.61 V) (Schrauzer et al. 1968, Schrauzer and Deutsch 1969, Banerjee et al. 1990). It oxidizes instantaneously in air and rapidly reduces protons to H2 gas at pH 7 (Schneider 1987). Hence, within the cells, sequestration of cob(I)alamin would be important by preventing nonspecific or deleterious side reactions. The finding that cob(I)alamin was sequestered during the conversion of cob(II)alamin to AdoCbl also indicates a physical interaction between the MSR and ATR. This raises the question of whether MSR interacts with both MS and ATR by a conserved mechanism. Prior studies of the MSR-MS reaction indicated that the optimal stoichiometry is ~4 MSR/MS and that activity is maximal over a narrow range of ionic strength indicating that electrostatic interactions are important to the MSR-MS interaction (Olteanu and Banerjee 2001). Here we found that the optimal ratio for the MSR-ATR system is also ~4 MSR/ATR, but in contrast to the MSR-MS system, there was no strict dependence on ionic strength. Hence, although MSR has a conserved function in both systems (cob(II)alamin reductase), the details of the protein-protein interactions between MSR and either MS or ATR are apparently somewhat different. The investigations reported here support prior studies that indicate redundant systems for mitochondrial cobalamin reduction in mammals. Several lines of evidence indicated a role for MSR in the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis: the MSR and ATR enzymes converted cob(II)alamin to AdoCbl at a significant
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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
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
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: Johnson et al. 2001, Saridakis et a
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
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129 Fenton, W. A. and L. E. Rosenbe
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131 Johnson, C. L. V. J., E. Pechon
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133 Mellman, I., H. F. Willard and
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135 Rossi, M., J. P. Glusker, L. Ra
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137 Vlasie, M., S. Chowdhury and R.
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BIOGRAPHICAL SKETCH Nicole Aurora L
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