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DE-SC0016510: Engineering a Functional Equivalent of Nitrogenase for Mechanistic Investigations of Ammonia Synthesis

Award Status: Active
  • Institution: Regents of the University of California, Irvine, Irvine, CA
  • DUNS: 046705849
  • PM: Stack, Robert
  • Most Recent Award Date: 06/29/2017
  • Number of Support Periods: 2
  • PI: Hu, Yilin
  • Current Budget Period: 09/15/2017 - 09/14/2018
  • Current Project Period: 09/15/2016 - 09/14/2019
  • Supplement Budget Period: N/A
 

Public Abstract

PROJECT SUMMARY/ABSTRACT

 

PROJECT TITLE

Engineering a Functional Equivalent of Nitrogenase for Mechanistic Investigations of Ammonia Synthesis

 

PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR (PD/PI)

Yilin Hu

Department of Molecular Biology & Biochemistry University of California, Irvine

 

CO-PRINCIPAL INVESTIGATOR (co-PI)

Markus Ribbe

Departments of Molecular Biology & Biochemistry, and Chemistry University of California, Irvine

 

COLLABORATORS

Professors Keith Hodgson and Britt Hedman

Stanford University, Menlo Park, CA (XAS/EXAFS Spectroscopy)

 

ABSTRACT

Nitrogenase catalyzes the conversion of inert dinitrogen to bioavailable ammonia. Despite major efforts in the past decades, the catalytic mechanism of nitrogenase has not been fully deciphered. Previous studies have shown that NifEN, a scaffold protein that hosts the biosynthesis of nitrogenase cofactor, is a functional homolog of MoFe protein (the catalytic component of nitrogenase). Compared to MoFe protein, NifEN has a lower enzymatic activity and a narrower substrate profile, making it a perfect mutational platform for (re)constructing a functional MoFe protein. This project aims at generating a functional MoFe protein equivalent by sequentially reconstructing a P-cluster site, sequentially reconstructing a P-cluster site (Aim 1), duplicating an M-cluster site (Aim 2) and re- establishing proton gating residues in NifEN (Aim 3); functional variants of nitrogenase with altered activity and/or product profile will be generated by mixing-and-matching the reconstructions of the key catalytic features in NifEN (Aim 4). Genetic methods will be used to systematically reconstruct an equivalent or variant of MoFe protein; and biochemical and spectroscopic methods will be employed to monitor and analyze the (re)construction process. Success in generating an active nitrogenase equivalent on a NifEN template will enable identification of all functional determinants for the catalytic activity of nitrogenase and provide a proof-of-concept for minimizing the essential nif gene set and engineering a metabolic pathway of nitrogenase assembly and function via a synthetic biology approach; whereas success in generating functional variants of nitrogenase will facilitate capture of substrates/intermediates or permit identification of candidates with desired product profile, both of which contribute to a better understanding of the mechanism of ammonia synthesis by nitrogenase. As such, the outcome of the proposed activities could prove instrumental in decoding the nitrogenase mechanism and enable development of energy-efficient strategies for sustainable ammonia synthesis.



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