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

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14 purification, explaining why cob(III)alamin reductase activity was retained without the addition of these flavins. Cell extracts from human fibroblasts and rat liver have cob(III)alamin reductase activity associated with both microsomes and the mitochondrial membrane, that was derived from oxidation-reduction reactions by cytochromes and flavoproteins (Watanabe et al. 1989, Watanabe et al. 1996). There has been no genetic evidence that the flavoproteins studied in these systems are the physiological cob(III)alamin reductases. The studies by Watanabe and colleagues also indicate that both prokaryotic and eukaryotic systems produce redundant enzymes with the ability to reduce cobalamin; however, this reduction could be a primarily nonenzymatic process. Cob(II)alamin reductase Cob(I)alamin is one of the strongest nucleophiles known to exist in aqueous solution, and is a powerful reductant (E°’ = -0.61 V) (Banerjee et al. 1990). Because of the nucleophilic nature of cob(I)alamin, cob(II)alamin reduction is an energetically unfavorable reaction. Thus, it is no surprise that cob(II)alamin reductase activity can only be studied by coupling the reaction to an alkylation event such as adenosylation or methylation. Cob(II)alamin reductase activity has been detected in cell-free extracts of C. tetanomorphum and P. shermanii; however, these enzymes have not been identified, and the encoding genes are unknown (Brady et al. 1962, Weissbach et al. 1962, Walker et al. 1969). In humans, cob(II)alamin reductase activity has been detected in mitochondrial fractions, but the enzyme was never identified, and the gene is unknown (Fenton and Rosenberg 1978b). Purification of the cob(II)alamin reductase from cell extracts of C. tetanomorphum did not produce a single enzyme, but produced a complex containing the adenosylating
enzyme. This suggests that cob(II)alamin reductase and the adenosyltransferase may 15 exist as a structural complex under certain conditions (Walker et al. 1969). Advantages of this complex would decrease the probability of unfavorable cob(I)alamin side reactions and instead allow for adenosylation because of the close proximity of cob(I)alamin to the adenosyltransferase. The E. coli FldA protein has been shown to function as a cob(II)alamin reductase for the formation of AdoCbl (Fonseca and Escalante-Semerena 2001). In vitro, flavodoxin reductase (Fpr), flavodoxin (FldA), and purified CobA (an adenosyltransferase) catalyze AdoCbl synthesis from cob(III)alamin (Fonseca and Escalante-Semerena 2001). In this coupled assay, it is proposed that Fpr uses NADPH to reduce FldA, which then reduces cob(III)alamin to cob(I)alamin while bound to CobA (Fonseca and Escalante-Semerena 2001). To date, there are no reports showing an interaction between FldA and CobA. Although this reducing system is functional in vitro, there is no genetic evidence that FldA is the physiological cob(II)alamin reductase (Fonseca and Escalante-Semerena 2001). Adenosyltransferase Adenosylation is the terminal step in AdoCbl metabolism. ATP:cob(I)alamin adenosyltransferase (ATR) activity was detected in crude cell extracts of human fibroblast cells, but the enzyme involved and the encoding gene were never identified (Fenton and Rosenberg 1981). ATRs from P shermanii, C. tetanomorphum, P. denitrificans, Thermoplasma acidophilum, and S. enterica have been purified and partially characterized (Brady et al. 1962, Vitols et al. 1966, Debussche et al. 1991, Suh and Escalante-Semerena 1995, Johnson et al. 2001, Saridakis et al. 2004). The genes encoding these enzymes have been identified from P. denitrificans, T. acidophilum, and
Page 1 and 2: B12 METABOLISM IN HUMANS By NICOLE
Page 3 and 4: The work in this dissertation is de
Page 5 and 6: TABLE OF CONTENTS Page ACKNOWLEDGME
Page 7 and 8: General Molecular and Protein Metho
Page 9 and 10: LIST OF TABLES Table page 1-1. Aden
Page 11 and 12: 3-7. Stoichiometry of the MSR-ATR s
Page 13 and 14: DTT dithiothreitol E. coli Escheric
Page 15 and 16: Abstract of Dissertation Presented
Page 17 and 18: CHAPTER 1 INTRODUCTION History of C
Page 19 and 20: 3 (DMB). The DMB moiety is covalent
Page 21 and 22: 5 and deoxyadenosine. After hydroge
Page 23 and 24: 7 homocysteine recycling and methio
Page 25 and 26: methylmalonic acid. Methylmalonic a
Page 27 and 28: folate-dependent reactions includin
Page 29: tetanomorphum, P. shermanii, Euglen
Page 33 and 34: at the 5’-C of ATP by cob(I)alami
Page 35 and 36: iochemical studies and complementat
Page 37 and 38: 1997). Both cases of the cblD disor
Page 39 and 40: 23 The cblB complementation group i
Page 41 and 42: (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 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 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 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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