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

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112 dehydrogenase (aldehyde-consuming) is consistent with the prior proposal that these structures function to minimize aldehyde toxicity (Chen et al. 1994, Stojiljkovic et al. 1995, Havemann et al. 2002). Presumably, the shell of the polyhedral body could trap the propionaldehyde produced by the PduCDE diol dehydratase until it is consumed by the PduP propionaldehyde dehydrogenase. Sequestering propionaldehyde until it is converted to propionyl-CoA might protect sensitive cytoplasmic components. Alternatively, the inclusion of diol dehydratase and propionaldehyde dehydrogenase in the pdu polyhedra might simply serve to juxtapose these enzymes in order to facilitate propionaldehyde channeling, although it is unclear why such a complex structure would be needed for this sole purpose. Prior studies suggested that the pdu polyhedra minimize aldehyde toxicity by limiting aldehyde production through control of AdoCbl availability (Havemann et al. 2002). Such a mechanism would be improved by either aldehyde channeling or sequestration, and a combined mechanism that fine tunes propionaldehyde production and consumption could explain the selective advantage provided by these complex polyhedral bodies involved in 1,2-propanediol degradation.
113 Table 4-1. Bacterial strains Species Strain Genotype E. coli BL21 (DE3) RIL BE237 BE273 (E. coli B) F - ompT hsdS (rB - mB - ) dcm + Tet r gal λ (DE3) endA Hte (argU ileY leuW Cam r ) BL21 (DE3) RIL/pET-41a (T7 expression vector without insert, Kan r ) BL21 (DE3) RIL/pNL69 (T7expression vector with insert encoding His8-PduP) S17.1λpir recA(RP4-2-Tc::Mu) λpir BE47 thr-480::Tn10dCam S. enterica serovar Typhimurium LT2 TR6579 metA22 metE551 trpD2 ilv-452 hsdLt6 hsdSA29 HsdB - strA120 GalE - Leu - Pro - BE191 ∆pduP659 BE268 LT2/ pNL9 (pLAC22-pduP, Ap r ) BE269 ∆pduP659/ pLAC22 (vector without insert, Ap r ) BE270 ∆pduP659/pNL9 (pLAC22-pduP, Ap r )
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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
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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: 111 biochemical evidence was presen
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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