Dataset Information

Nitrogenase Fe protein: A molybdate/homocitrate insertase.

ABSTRACT: The Fe protein is indispensable for nitrogenase catalysis and biosynthesis. However, its function in iron-molybdenum cofactor (FeMoco) biosynthesis has not been clearly defined. Here we show that the Fe protein can act as a Mo/homocitrate insertase that mobilizes Mo/homocitrate for the maturation of FeMoco precursor on NifEN. Further, we establish that Mo/homocitrate mobilization by the Fe protein likely involves hydrolysis of MgATP and protein-protein interaction between the Fe protein and NifEN. Our findings not only clarify the role of the Fe protein in FeMoco assembly and assign another function to this multitask enzyme but also provide useful insights into a mechanism of metal trafficking required for the assembly of complex metalloproteins such as nitrogenase.

SUBMITTER: Hu Y

PROVIDER: S-EPMC1859896 | biostudies-other | 2006 Nov

REPOSITORIES: biostudies-other

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Publications

Nitrogenase Fe protein: A molybdate/homocitrate insertase.

Hu Yilin Y Corbett Mary C MC Fay Aaron W AW Webber Jerome A JA Hodgson Keith O KO Hedman Britt B Ribbe Markus W MW

Proceedings of the National Academy of Sciences of the United States of America 20061024 46

The Fe protein is indispensable for nitrogenase catalysis and biosynthesis. However, its function in iron-molybdenum cofactor (FeMoco) biosynthesis has not been clearly defined. Here we show that the Fe protein can act as a Mo/homocitrate insertase that mobilizes Mo/homocitrate for the maturation of FeMoco precursor on NifEN. Further, we establish that Mo/homocitrate mobilization by the Fe protein likely involves hydrolysis of MgATP and protein-protein interaction between the Fe protein and NifE ...[more]

PMID: 17062756

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Project description:The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii (AvNifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli. Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed AvNifH protein has a high [Fe4S4] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that AvNifM could serve as a chaperone and assist the maturation of a cluster-replete AvNifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

Dataset Information

Nitrogenase Fe protein: A molybdate/homocitrate insertase.

Publications

Nitrogenase Fe protein: A molybdate/homocitrate insertase.

Similar Datasets

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