In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis
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© Springer-Verlag 1997
O R I G I N A L PA P E R
Sagar Sengupta · Melkote Subbarao Shaila · Gannamani Ramananda Rao
In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis
Received: 9 January 1997 / Accepted: 15 May 1997
Abstract The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the pathway, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis. Key words Candida utilis · Nitrite reductase · Electron donors · Cysteine modification · Transcriptional control
S. Sengupta (Y)1 · M. S. Shaila · G. R. Rao Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India Present address: 1 IGBMC, 1 Rue Laurent Fries, B.P. 163-67404, Illkirch Cedex, C.U. de Strasbourg, France Tel. +33-3-88-653402; Fax +33-3-88-653246 e-mail: [email protected]
Introduction Many bacteria, fungi, algae and yeasts obtain a major part of their nitrogen requirement from nitrate (Solomon and Vennesland 1972; Hewitt and Hucklesby 1966; Choudary and Rao 1984; Arizmendi and Serra 1990). The regulation of this assimilation pathway is, in turn, controlled by the activities of two enzymes, namely nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1), which catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonium, respectively. Nitrite reductases isolated from fungal and bacterial (i.e. non-photosynthetic) systems utilise either NADPH or NADH or both as the electron donor under physiological conditions. The purified enzymes from some prokaryotic organisms use only NADH as the electron donor (Coleman et al. 1978; Campbell and Kinghorn 1990). For nitrite reductase from Azotobacter chroococcum and Achromobacter fischeri, NADPH is a relatively ineffective
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