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

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52 WG2127 did not express detectable levels of the 25 kDa form of the ATR and expressed reduced levels of the 23 kDa form (lanes 2 and 4). For cblB mutant WG1879, the 25 kDa form was reduced, and the 23 kDa form was not detectable. These results indicated that expression of the ATR was significantly altered in cblB mutant human skin fibroblasts. These results provide direct evidence that defects in the ATR identified in this study underlie cblB methylmalonic aciduria. Conserved Amino Acids and Distribution of Adenosyltransferase Enzymes The human and bovine ATR enzymes were aligned with 20 related sequences obtained from GenBank (Figure 2-1). Of the sequences shown, the function of the H. sapiens, B. taurus, and S. enterica ATRs is supported by biochemical evidence presented here and previously (Johnson et al. 2001). To obtain additional information about the function of the remaining prokaryotic ATRs, we examined their genomic context. Genes encoding ATR homologues from S. enterica, L. innocua, L. monocytogenes, B. halodurans, C. perfringes, C. pasteurianum, and K. pneumonia were found proximal to either AdoCbl-dependent enzymes or AdoCbl biosynthetic genes. Since bacteria frequently cluster genes of related function, these finding suggest that the genes from these organisms do indeed encode ATR enzymes. It is also notable that there are several regions of high conservation in all the ATRs aligned suggesting that many are indeed ATR enzymes (Figure 2-1). Thus, PduO-type ATR enzymes appear to have broad phylogenetic distribution, being found in mammals, gram positive and gram negative bacteria, and the archaea. The highly conserved regions found in the ATRs also indicate amino acids likely to be essential for enzymatic activity, and may represent sites directly involved in catalysis or in binding of substrates, ATP and cob(I)alamin. There are at least two different ways
in which proteins bind B12, the "base-on" and "base-off" modes. Base-off binding 53 involves displacement of the lower axial ligand of B12 (dimethylbenzimidazole) with an imidazole side-chain of a histidine residue (Drennan et al. 1994, Shibata et al. 1999, Marsh and Drennan 2001). The sequence containing the coordinating histidine (D-X-H-X-X-G), which is conserved among proteins that bind B12 in the “base-off” mode, is absent from PduO-type ATRs examined in this study. This suggests that the ATR enzymes described in this study bind B12 in the “base-on” mode. Discussion In this report we identified human and bovine cDNAs that encode ATR enzymes. The highest specific activities measured in cell extracts from expression strains were 85.7 nmol min -1 mg -1 protein for the bovine ATR and 98 nmol min -1 mg -1 protein for the human ATR. These values are comparable to those previously reported for purified CobA ATR, and partially purified PduO ATR which were 53 and 312 nmol min -1 mg -1 protein, respectively (Suh and Escalante-Semerena 1993, Suh and Escalante-Semerena 1995). The bovine ATR cDNA was isolated based on its ability to complement an ATR-deficient S. enterica mutant for color formation on indicator medium, and the human ATR cDNA was shown to restore the ability of an ATR-deficient mutant to grow on 1,2-propanediol in an AdoCbl-dependent fashion. This demonstrated that the human and bovine cDNAs described here encode ATR enzymes that are active under physiological conditions, although in a heterologous host. Moreover, the bovine and human ATR cDNAs had 29% and 26% identity to the PduO ATR of S. enterica (Johnson et al. 2001) and the human enzyme included a presumptive MTS as expected (Fenton et al. 2000). Thus, genetic, biochemical, and bioinformatic evidence indicate that the bovine and human cDNAs analyzed here encode ATR enzymes.
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B12 METABOLISM IN HUMANS By NICOLE
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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 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: 51 of the lacI repressor (not shown
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
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111 biochemical evidence was presen
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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
Page 145 and 146:
129 Fenton, W. A. and L. E. Rosenbe
Page 147 and 148:
131 Johnson, C. L. V. J., E. Pechon
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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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