Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic sub unit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs front that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required mgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.

Building business cases for maintenance improvement projects

Many maintenance managers find it difficult to justify investments in maintenance improvement initiatives. In part, this is due to a tendency by mine managers to regard maintenance purely as a cost centre, and not as a process able to influence productive capacity and profit. It is also hindered by a lack of alignment between commonly used maintenance performance measures and key business drivers, and the lack of formal business training amongst maintenance professionals. With this in mind, a model to assist maintenance managers in evaluating the benefits of maintenance improvement projects was recently formulated. The model considers four cost saving dimensions. These are: 1. reduction in the cost of unplanned repairs and maintenance, 2. increased or accelerated production and/or sales, 3. spares inventory reduction, and 4. reduction in over-investment in physical assets and operating costs. This paper discusses the application of this model and a number of numerical examples are given to justify investments in maintenance improvement projects having varying objectives.