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

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134 Paschen, S. A. and W. Neupert. 2001. Protein import into mitochondria. IUBMB Life 52:101-12. Pezacka, E. 1993. Identification of and characterization of two enzymes involved in the intracellular metabolism of cobalamin. Cyanocobalamin β-ligand transferase and micosomal cob(III)alamin reductase. Biochem. et. Biophys. Acta. 1157:167-177. Pezacka, E., R. Green and D. W. Jacobsen. 1990. Glutathionylcobalamin as an intermediate in the formation of cobalamin coenzymes. Biochem. Biophys. Res. Commun. 169:443-450. Price, G. D., S. Maeda, T. Omata and M. R. Badger. 2002. Modes of active inorganic carbon uptake in the cyanobacterium, Synechococcus sp PCC7942. Functional Plant Biology 29:131-149. Price-Carter, M., J. Tingey, T. A. Bobik and J. R. Roth. 2001. The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar typhimurium on ethanolamine or 1,2-propanediol. J. Bacteriol. 183:2463-75. Qureshi, A. A., D. S. Rosenblatt and B. A. Cooper. 1994. Inherited disorders of cobalamin metabolism. Crit. Rev. Oncol. Hematol. 17:133-51. Refsum, H., P. M. Ueland, O. Nygard and S. E. Vollset. 1998. Homocysteine and cardiovascular disease. Annu. Rev. Med. 49:31-62. Reichard, P. 1993. The anaerobic ribonucleotide reductase from Escherichia coli. J. Biol. Chem. 268:8383-6. Rickes, E. L., N. G. Brink, F. R. Koniuszy, T. R. Wood and K. Folkers. 1948. Crystalline vitamin B12. Science 107:396-397. Rondon, M. R., R. Kazmierczak and J. C. Escalante-Semerena. 1995. Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2. J. Bacteriol. 177:5434-9. Rosenblatt, D. S., A. L. Aspler, M. I. Shevell, B. A. Pletcher, W. A. Fenton and M. R. Seashore. 1997. Clinical heterogeneity and prognosis in combined methylmalonic aciduria and homocystinuria (cblC). J. Inherit. Metab. Dis. 20:528-38. Rosenblatt, D. S. and A. B. Cooper. 1990. Inherited disorders of vitamin B12 utilization. Bioessays 12:331-334. Rosenblatt, D. S. and W. A. Fenton. 1999. Inborn errors of cobalamin metabolism, p. 367-384. In R. Banerjee (ed.), Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York.
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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
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Linearity of the ATR reaction 71 Th
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73 reactions was characteristic of
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75 MSR Produces little Cob(I)alamin
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Johnson et al. 2001, Saridakis et a
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AdoCbl by the MSR-ATR system. Exper
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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: 133 Mellman, I., H. F. Willard and
Page 153 and 154: 137 Vlasie, M., S. Chowdhury and R.
Page 155 and 156: BIOGRAPHICAL SKETCH Nicole Aurora L
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