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

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104 about 7.4 and 21.7 hours, respectively, and reached maximum optical densities at 600 nm of about 1.38 and 0.49, respectively. This showed the ∆pduP mutant was significantly impaired for growth on 1,2-propanediol/CNCbl minimal medium. As a control, we tested whether the growth defect of strain BE191 (∆pduP) could be corrected by the expression of pduP in trans. The generation times of strains BE270 (∆pduP/pLAC22-pduP) and BE269 (∆pduP/pLAC22-no insert) on 1,2-propanediol/CNCbl minimal medium were 3.2 and 25.4 hours, respectively, and the maximum optical densities reached by these strains were 1.7 for BE270 and 0.45 for BE269. Hence, expression of pduP in trans corrected the growth defect of ∆pduP mutation on 1,2-propanediol/CNCbl minimal medium. This result indicated that the observed growth defect of BE191 was due to deletion of pduP, but not to polarity or a mutation inadvertently introduced during strain construction. This was the expected result since BE191 was constructed by a PCR-based method designed to produce a precise nonpolar deletion. Strain BE270 was found to grow about 2.2-fold faster than did the wild-type strain. It appeared that pLAC22 enhanced growth of this strain on 1,2-propanediol minimal medium for unknown reasons, since the wild-type strain also grew faster when it carried this vector without insert (not shown). Growth tests were repeated twice with similar results. The fact that BE191 (∆pduP) was impaired for growth on 1,2-propanediol/CNCbl minimal medium, in conjunction with the control experiments described above indicates that pduP plays an important role in 1,2-propanediol degradation in vivo.
105 Propionaldehyde Dehydrogenase Activity in Wild-type S. enterica and a ∆pduP Mutant To test whether the pduP gene encodes a propionaldehyde dehydrogenase, enzyme assays were performed on cell extracts of wild-type S. enterica and strain BE191 (∆pduP). The ∆pduP mutant used was the same strain used for the growth studies described above, and assays were conducted on both the supernatants and the pellets that resulted from centrifugation of cell extracts at 31,000 x g (Table 4-2). For the wild-type strain, the majority of the propionaldehyde dehydrogenase activity was found in the 31,000 x g pellet which had a specific activity 1.15 µmol min -1 mg -1 protein. Similar extracts from the pduP mutant had only 1% of the specific activity of the wild-type strain (0.011 µmol min -1 mg -1 ). When the substrates, propionaldehyde, NAD + or HS-CoA were omitted from the assay mixture, no activity was detected. To our knowledge, this was the first experimental evidence that the pduP gene encodes a CoA-acylating propionaldehyde dehydrogenase. It was of interest that the majority of propionaldehyde dehydrogenase activity was found in the 31,000 x g pellet from the wild-type strain (99%). Prior studies showed that polyhedral bodies are involved in AdoCbl-dependent 1,2-propanediol degradation by S. enterica (Bobik et al. 1999, Havemann et al. 2002). Hence, the finding that the PduP aldehyde dehydrogenase was associated with the 31,000 x g pellet suggested that it might be associated with the polyhedral bodies which pellet under similar centrifugation conditions (unpublished results). High-level Production of the PduP Protein E. coli strain BE273 (pET41a-His8-pduP) was constructed to produce high levels of recombinant His8-PduP protein. Protein production by this strain as well as by the
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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
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: 103 from New England Biolabs (Bever
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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