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

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2 laboratory of Dorothy Hodgkin (Hodgkin et al. 1955), a feat for which she was awarded the 1964 Nobel Prize in Chemistry. Today we know that two active cobalamin derivatives are required for enzyme catalysis. One is the adenosyl derivative (adenosylcobalamin) which was identified in 1958, when Barker and coworkers reported the isolation of a light-sensitive cobalamin compound that was a required cofactor for glutamate mutase (Barker et al. 1958). Shortly after the discovery of this compound, its crystal structure was resolved by Lenhert and Hodgkin (Lenhert and Hodgkin 1961). The other biologically active cobalamin derivative was discovered later as methylcobalamin, a required cofactor for methionine synthase (Guest et al. 1962a, Guest et al. 1962b). The structure of methylcobalamin was resolved decades later by Jenny Gluskers laboratory (Rossi et al. 1985). Structure of Cobalamin Cobalamin is the largest and most complex cofactor known to man. It is a water-soluble molecule composed of three general structural units: a central ring, an upper ligand, and a lower ligand (Figure 1-1). At the core of cobalamin is a ring structure designated as corrin, which is synthesized from uroporphyrinogen III, the last common intermediate of heme, siroheme, chlorophyll, and cobalamin synthesis. The corrin ring consists of four reduced pyrrole rings (A, B, C, and D). Unlike heme and chlorophyll (in which the pyrrole rings are linked by four methylene bridges), cobalamin has a direct bond between rings A and D. In cobalamin, the four nitrogen atoms of the pyrrole rings form coordinate bonds with a central cobalt atom; whereas the central atom of the tetrapyrrole ring is iron and magnesium, in heme and chlorophyll, respectively. Below the plane of the corrin ring is the α-ligand of the cobalt atom, dimethylbenzimidazole
3 (DMB). The DMB moiety is covalently attached to the corrin ring by a ribose phosphate bonded to the aminopropanol side chain on ring D and is also coordinated to the central cobalt atom. The exact function of the lower ligand is not completely understood, but has been suggested that it may have a role in coenzyme binding and catalysis (Banerjee and Ragsdale 2003). Above the plane of the corrin ring is the β-ligand of the cobalt atom. The β-ligand varies in the different forms of cobalamins and includes cyano, hydroxy, glutathionyl, methyl, and adenosyl moieties. Cyanocobalamin (vitamin B12, CNCbl) is an artifact obtained during isolation of cobalamins from natural sources and is the most common pharmaceutical form (Rickes et al. 1948). CNCbl together with hydroxycobalamin (HOCbl), and glutathionylcobalamin (GSCbl) are precursors for synthesis of the biologically active coenzymes, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl). Biosynthesis of Cobalamin Cobalamin or closely related corrinoid compounds are required in all kingdoms of life with perhaps the exception of plants and fungi. De novo synthesis of B12 is restricted to some Bacteria and Archaea (Roth et al. 1996), requiring all other life forms that use B12 to obtain complex precursors from their diet. Characterized as the most complex nonpolymeric natural substance known, cobalamin is synthesized de novo by a multi-step enzymatic process consisting of uroporphyrinogen III synthesis, side chain modification, cobalt insertion, aminopropanol addition, DMB biosynthesis, and addition of methyl or adenosyl moieties (Battersby 1994). The microbial biosynthesis of cobalamin can proceed along two different pathways: one aerobic and the other anaerobic (Scott 2003). These pathways differ both in their requirement for molecular oxygen and the timing of
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: CHAPTER 1 INTRODUCTION History of C
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 and 30: tetanomorphum, P. shermanii, Euglen
Page 31 and 32: enzyme. This suggests that cob(II)a
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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