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Abstract
Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.
Highlights
Dissimilatory nitrate reductase A from Escherichia coli (NarGHI) is one of the best studied multisubunit molybdoenzymes [14] and can be considered as a model system for studying the biogenesis process in prokaryotic enzymes
In the case of the nitrate reductase from T. thermophilus, it has been demonstrated that NarJ-assisted molybdenum cofactor (Moco) incorporation occurs within the membranebound apoenzyme complex
The work presented here has answered two fundamental questions on molybdoenzyme biogenesis. (i) Considering the multisubunit character of numerous molybdoenzymes, at which particular stage of assembly does the Moco insertion event occur? (ii) How could such an event be controlled? Here, using the heterotrimeric nitrate reductase A from E. coli as a model, we demonstrated that the enzyme-specific chaperone NarJ binds two distinct sites on the apoenzyme to coordinate both molybdenum cofactor insertion and multisubunit assembly
Summary
Dissimilatory nitrate reductase A from Escherichia coli (NarGHI) is one of the best studied multisubunit molybdoenzymes [14] and can be considered as a model system for studying the biogenesis process in prokaryotic enzymes. The enzyme-specific chaperone NarJ plays an essential role for nitrate reductase A activity, facilitating Moco insertion into NarG [11]. As observed for other known molybdoenzymes (16 –19), the crystal structure of the NarGHI complex [20, 21] reveals that Moco is an extended molecule deeply buried into the enzyme complex at the NarG-H subunit interface. This observation suggests that the subunit assembly and the Moco incorporation must be tightly coordinated. The enzyme-specific NarJ protein coordinates assembly and molybdenum cofactor acquisition of the heterotrimeric enzyme during the biogenesis process
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