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

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110 polyhedra is more easily discerned). This result indicated that PduP is nonessential for polyhedral body formation. Propionaldehyde Dehydrogenase Activity is associated with Purified pdu Bodies To further investigate the subcellular localization of the PduP enzyme, the polyhedral bodies involved in 1,2-propanediol degradation were purified and propionaldehyde dehydrogenase activity was followed during the course of their purification (Table 4-3). The polyhedra were purified by a combination of detergent treatment and differential and density-gradient centrifugation as described in Havemann and Bobik (2003). Electron microscopy and SDS-PAGE indicated that the polyhedra obtained were highly purified (not shown). During the purification the specific activity of the PduP propionaldehyde dehydrogenase increased 13.5-fold from 0.3 - 4.0 µmol min -1 mg -1 protein (Table 4-3). This suggested that the polyhedral bodies comprised about 7% of the total cell protein, which is consistent with previous electron microscopy (Havemann et al. 2002). Furthermore, Western blots verified that the PduP enzyme was associated with the purified polyhedra (not shown). Thus, enzyme assays and Western blots with purified polyhedra supported the immunolabeling studies described above and indicated that the PduP propionaldehyde dehydrogenase is associated with the polyhedral bodies involved in 1,2-propanediol degradation. Discussion Prior biochemical studies indicated that a CoA-acylating propionaldehyde dehydrogenase was required for AdoCbl-dependent 1,2-propanediol degradation (Toraya et al. 1979, Obradors et al. 1988). Subsequent DNA sequence analyses of the pdu operon identified a presumptive enzyme (PduP) that was 32% identical to the E. coli aldehyde/alcohol dehydrogenase, AdhE (Bobik et al. 1999). In this report, genetic and
111 biochemical evidence was presented that established PduP as a CoA-acylating propionaldehyde dehydrogenase involved in AdoCbl-dependent 1,2-propanediol degradation by S. enterica. Previously, Jorge Escalante's group proposed that two propionaldehyde dehydrogenases might be involved in 1,2-propanediol degradation by S. enterica: one that produces propionyl-CoA and a second that oxidizes propionaldehyde directly to propionate (Palacios et al. 2003). The PduP enzyme studied here produced propionyl-CoA as a reaction product and was inactive in the absence of HS-CoA showing that it is incapable of oxidizing propionaldehyde directly to propionate under the assay conditions used. It was also shown that an S. enterica strain with a nonpolar pduP null mutation grew at one-third the rate of the wild-type strain. The residual growth of a ∆pduP mutant could have resulted from the activity of an alternative propionaldehyde dehydrogenase, such as the one proposed to convert propionaldehyde directly to propionate (Palacios et al. 2003), since the propionate produced by this enzyme could be used as a carbon and energy source via the methyl citrate pathway (Horswill and Escalante-Semerena 1997). Previously, we established that S. enterica formed polyhedral bodies during AdoCbl-dependent growth on 1,2-propanediol (Bobik et al. 1999). Immunolabeling studies demonstrated that these bodies consisted of a protein shell (partly composed of the PduA protein) AdoCbl-dependent diol dehydratase and additional unidentified proteins (Bobik et al. 1999, Havemann et al. 2002). In this report, we presented several lines of evidence that indicated PduP is polyhedral-body-associated. The finding that the pdu polyhedra include both diol dehydratase (aldehyde-producing) and propionaldehyde
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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
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: 109 antiserum obtained was specific
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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