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

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64 to the genes for MSR (MTRR) and MS (MTR), respectively, and defects in either gene results in homocystinuria (Cooper and Rosenblatt 1987, Watkins and Rosenblatt 2001). The mut group corresponds to the gene that encodes MCM (MUT). The cblA group (MMAA gene) encodes a protein proposed to protect MCM from inactivation (Korotkova and Lidstrom 2004) and recent studies have shown that the cblB complementation group (MMAB gene) encodes a mitochondrial ATP cob(I)alamin adenosyltransferase (ATR) (Dobson et al. 2002, Leal et al. 2003). Mutations in the cblH complementation group also result in methylmalonic aciduria, but the nature of the underlying defect and the corresponding gene are unknown (Watkins and Rosenblatt 2001). The human enzyme that catalyzes the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis has not been identified. Recent studies showed that MSR mediates cob(II)alamin reduction for MS activation (Leclerc et al. 1999, Olteanu and Banerjee 2001), raising the possibility that this enzyme might play a role in AdoCbl synthesis. Here we describe the purification and initial biochemical characterization of the human ATR from recombinant E. coli and demonstrate that in vitro human MSR can reduce cob(II)alamin to cob(I)alamin for AdoCbl synthesis by the ATR enzyme. Chemicals and Reagents Materials and Methods Restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA). Titanium III citrate was prepared as previously described (Bobik and Wolfe 1989). AdoCbl, HOCbl, β-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), iodoacetic acid (IA), flavin adenine dinucleotide (FAD), and bovine serum albumin (BSA) were purchased from Sigma Chemical Company (St. Louis, MO). Isopropyl-ß-D-thiogalactopyranoside (IPTG), ultra-pure ammonium sulfate, adenosine
65 5’- triphosphate (ATP), and dithiothreitol (DTT) were purchased from ICN Biomedicals, Inc. (Aurora, Ohio). Pefabloc SC PLUS was from Roche Diagnostics (Penzberg, Germany). All other chemicals and reagents were from Fisher Scientific (Norcross, GA). Bacterial Strains and Growth Media The bacterial strains used were E. coli DH5α and BL21 (DE3) RIL (Stratagene, La Jolla, CA). LB was the rich medium used (Difco, Detroit, MI) (Miller 1972). LB was supplemented with 25 µg/ml kanamycin and 20 µg/ml chloramphenicol or as indicated. General Molecular and Protein Methods Agarose gel electrophoresis and restriction enzyme digests were performed using standard protocols (Maniatis et al. 1982, Johnson et al. 2001). PCR products and plasmid DNA were gel purified using QIAquick ® gel extraction kit (QIAGEN, Inc., Valencia, CA). DNA ligation was accomplished using T4 DNA ligase according to manufacturer’s instructions. Bacterial transformation and SDS-PAGE were performed as previously described (Johnson et al. 2001). ATP:cob(I)alamin Adenosyltransferase Assays ATR assays were performed as previously reported with some modifications (Johnson et al. 2001). Reaction mixtures contained: 200 mM Tris-HCl (pH 8.0), 2.8 mM MgCl2, 10 mM KCl, 0.05 mM HOCbl, 0.4 mM ATP, and 1 mM titanium(III) citrate in a total volume of 2 ml. Reaction components (except for the ATR) were dispensed into cuvettes inside an anaerobic chamber (Coy Laboratories, Ann Arbor, MI). The cuvettes were sealed, removed from the chamber, and incubated at 37°C for 2 minutes. Reactions were initiated by addition of purified recombinant ATR, and AdoCbl formation was measured by following the decrease in absorbance at 388 nm (∆ε388 = 24.9 cm -1 mM -1 ).
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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
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
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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: 63 (cyanocobalamin, CNCbl) and hydr
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
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115 Table 4-3. Propionaldehyde dehy
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kDa 209 80 49 35 29 117 1 2 3 4 Fig
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CHAPTER 5 CONCLUSIONS In humans, co
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121 substitutions that are common p
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123 Studies also indicated that hum
Page 141 and 142:
LIST OF REFERENCES Aberg, A., S. Ha
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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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