MOLYBDENUM COFACTOR REQUIREMENT FOR INVITRO ACTIVATION OF APO-MOLYBDOENZYMES OF ESCHERICHIA-COLI

The apo‐nitrate reductase precursor in an Escherichia coli chiB mutant preparation obtained following growth in the presence of tungstate is activated by incubation with protein FA and a heat‐treated preparation from an E. coli crude extract. We show that the requirement for heat‐treated E. coli crude extract can be fulfilled by material obtained from either of two heat–denatured purified E. coli molybdoenzymes, namely nitrate reductase or trimethylamine N‐oxide reductase. Apo‐trimethylamine N‐oxide reductase precursor in the tungstate‐grown chIB preparation can be activated in a similar manner with material from either heat‐denatured molybdoenzyme. The active component in the denatured molybdoenzyme preparations is shown to be the molybdenum cofactor by Neurospora crassa nit1 molybdenum cofactor assay, size estimation and fluorimetric analysis. The direct demonstration of the requirement for molybdenum cofactor in the E. coli tungstate‐grown chIB complementation system is an important step towards the molecular definition of the activation process and an understanding of the mechanism of cofactor acquisition during molybdoenzyme biosynthesis. Copyright © 1990, Wiley Blackwell. All rights reserved