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

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80 rate; the stoichiometry needed for maximal activity was reasonable (4 MSR/ATR); and cob(I)alamin was sequestered as is expected for the physiologically relevant system. On the other hand, prior investigations showed that patients with inherited defects in MSR (cblE disorder) generally have homocystinuria with the exception of one reported case resulting in combined homocystinuria and mild methylmalonic aciduria (Wilson et al. 1999). This indicates that in vivo cob(II)alamin reduction for AdoCbl synthesis can occur independently of MSR. Of the known complementation groups that result in methylmalonic aciduria, only the cblH and cblA groups have phenotypes consistent with a role in mitochondrial cobalamin reduction. Recent studies have indicated that the cblA group functions to protect MCM from inactivation (Korotkova and Lidstrom 2004) and there has only been one reported case of cblH methylmalonic aciduria (Watkins et al. 2000, Dobson et al. 2002). Thus at this time, there are no strong indications as to the identity of the alternative reductase(s). Redundant systems for cobalamin metabolism are known in bacterial systems (Fonseca and Escalante-Semerena 2000, Johnson et al. 2001), and unpublished results). There, inducible systems provide extra capacity during times of high demand. Similarly, in humans multiple cobalamin reductases might be required for an efficient response to physiological stresses of diets that result in higher rates of propionyl-CoA formation. For example, MSR could be targeted to the mitochondria under circumstances where the diet is rich in amino acids metabolized via propionyl-CoA.
propionyl-CoA PCC (2S)-methylmalonyl-CoA MCEE (2R)-methylmalonyl-CoA MCM succinyl-CoA CENTRAL <strong>METABOLISM</strong> breakdown breakdown (AdoCbl (AdoCbl is is unstable unstable in in vivo) vivo) β-ligand transferase 81 CNCbl GSCbl cob(III)alamin reductase cob(II)alamin MSR cob(I)alamin ATR AdoCbl Methionine synthase [cob(II)alamin] MSR CH CH3THF 3THF + homocysteine Methionine synthase [CH [CH3Cbl] 3Cbl] inactivation inactivation THF + methionine Figure 3-1. Propionyl-CoA metabolism, methionine synthesis, and intracellular cobalamin metabolism. In humans, the pathways shown are needed for the complete catabolism of compounds degraded via propionyl-CoA and for recycling homocysteine. Abbreviations: PPC, propionyl-CoA carboxylase; MCEE, methylmalonyl-CoA epimerase; MCM, AdoCbl-dependent methylmalonyl-CoA mutase; CNCbl, vitamin <strong>B12</strong>; GSCbl, glutathionylcobalamin, MSR, methionine synthase reductase; ATR, ATP:cob(I)alamin adenosyltransferase; THF, tetrahydrofolate.
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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
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 and 94: Johnson et al. 2001, Saridakis et a
Page 95: AdoCbl by the MSR-ATR system. Exper
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
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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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