Membrane-Associated Maturation of the Heterotetrameric Nitrate Reductase ofThermus thermophilus

Abstract

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.

NarJ Chaperone Binds on Two Distinct Sites of the Aponitrate Reductase of Escherichia coli to Coordinate Molybdenum Cofactor Insertion and Assembly

“…Such coordination has been shown using the membrane-bound nitrate reductase from Thermus thermophilus. Indeed, the NarJ-assisted Moco insertion step can only occur once the apoenzyme complex is attached to the cytoplasmic membrane (22).In this work, we have provided new insights about molybdoenzyme biogenesis in E. coli. We have revealed a novel function for the enzymespecific chaperone NarJ that goes beyond its reported implication in the Moco incorporation process.…”

“…Recently, studies on the membrane-bound nitrate reductase (NarCGHI) from T. thermophilus revealed that multisubunit assembly and Moco incorporation are coupled (22). However, a different biogenesis pathway has been delineated as compared with the E. coli enzyme.…”

“…However, a different biogenesis pathway has been delineated as compared with the E. coli enzyme. Indeed, the NarJassisted Moco incorporation step strictly requires prior membrane anchoring of the apoNarGH complex to the NarCI anchor subunits (22). Further, sequence analysis of the NarG subunit from T. thermophilus revealed the absence of the N-terminal tail targeted by NarJ in the E. coli NarG protein.…”

“…In the case of the nitrate reductase from T. thermophilus, it has been demonstrated that NarJ-assisted Moco incorporation occurs within the membranebound apoenzyme complex. Further, NarJ is required for membrane attachment of the thermophilic apoenzyme complex (22). Thus, we investigated whether the cellular distribution of the apoenzyme from E. coli is influenced by its interaction with NarJ ( Fig.…”

“…Such coordination has been shown using the membrane-bound nitrate reductase from Thermus thermophilus. Indeed, the NarJ-assisted Moco insertion step can only occur once the apoenzyme complex is attached to the cytoplasmic membrane (22).…”