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Title: Biochemistry and molecular biology of amidases from Methylophilus methylotrophus.
Authors: Wyborn, Neil Ross.
Award date: 1994
Presented at: University of Leicester
Abstract: The biochemistry and molecular biology of amidases from M. methylotrophus was investigated. Acetamidase purification from whole cells exhibiting low specific activities yielded pure low-activity acetamidase (specific activity 6-15 umol min-1 [mg protein]-1), the activity of which could be reactivated to a level approaching that of the high-activity acetamidase by heating (1-6 h, 60 °C) with an activator component. Identical purifications from whole cells with high specific activities produced pure 'high-activity' acetamidases exhibiting a wide range of generally diminished activities (19-108 umol min-1 [mg protein]-1) and an unexpected propensity for heat-reactivation similar to that of low-activity acetamidase. It was concluded that high-activity acetamidases underwent varying degrees of 'switch-off of activity both pre- and post-purification. The physico-chemical properties of purified acetamidases were investigated in vitro to elucidate the nature of the putative acetamidase post-transcriptional modification and its role in the reversible regulation of acetamidase activity. High- and low-activity acetamidases exhibited significantly different properties, although their respective MW values differed only by 52Da. Low-activity acetamidase was significantly more stable than high-activity acetamidases which were labile. Results suggested that high- and low-activity acetamidases existed in different conformational states and that the regulation of acetamidase activity probably involved an allosteric mechanism. Cloning of the M. methylotrophus acetamidase structural gene (amiE) was unsuccessful, but the formamidase structural gene (fmd) was successfully cloned and heterologously- expressed in E. coli. The DNA sequences of fmd and three putative ORFs showed that (i) the formamidase primary sequence exhibited 57% strict identity with that of the Mycobacterium smegmatis 'acetamidase' (Mahenthiralingam et al., 1993), and (ii) ORF2 and ORF3 apparently respectively encoded a zinc finger DNA-binding protein and an AmiC-type regulatory protein. The evolutionary and regulatory implications of these findings were discussed.
Level: Doctoral
Qualification: Ph.D.
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Biochemistry
Leicester Theses

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