Abstract

Nitrogenases carry out the conversion of N2 to ammonium, a form of N that is more readily available for human nutrition. At the active site of nif-encoded, molybdenum nitrogenase is the iron-molybdenum cofactor, FeMo-co. The vnf-encoded nitrogenase contains the structurally similar iron-vanadium cofactor, FeV-co. The overall goal of this project is to determine the pathways of biosynthesis of FeMo-co and FeV-co in the nitrogen-fixing procaryote, Azotobacter vinelandii. The nifBUSV gene products are required for synthesis of both cofactors. In addition, the nifNE, nifH and nifX products are required for the synthesis of FeMo-co. When cells are grown on V in place of Mo, the cell synthesizes vnfNE, vnfH and vnfX gene products for the synthesis of FeV-co. The specific goals of this project are to define the roles of these gene products in the syntheses of FeMo-co/FeV-co and to establish the basis for the specificity for Mo or V during synthesis of each cofactor. NifB-co, the metabolic product of NifB, is an Fe and S donor to FeMo-co and FeV-co; the determination of the structure of NifB-co is a goal of this project. Several proteins in the FeMo-co synthesis pathway including NifH accumulate label from 55Fe-NifB-co or from 99MoO42- during the in vitro synthesis of FeMo-co, and 55Fe and 99Mo will be used to follow the steps in the pathway. Likewise, 49V accumulates on several proteins during FeV-co synthesis, and 55Fe and 49V will be used to follow the steps of the FeV-co pathway. VnfX has recently been identified as a protein that accumulates V and Fe during FeV-co synthesis, and the investigation of the role of VnfX in FeV-co synthesis will be a major goal of the project. All organisms require trace elements, and the information gained in this project will increase our understanding of the mechanisms by which living cells assimilate, accumulate, and discriminate between trace elements that are used to build complex cofactors required for many redox reactions crucial to the cell. Human health is dependent on proper metabolism and utilization of trace elements such as those being investigated in this study.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM035332-15
Application #
6329664
Study Section
Special Emphasis Panel (ZRG5-MBC-2 (01))
Program Officer
Ikeda, Richard A
Project Start
1986-12-01
Project End
2003-11-30
Budget Start
2000-12-01
Budget End
2001-11-30
Support Year
15
Fiscal Year
2001
Total Cost
$299,906
Indirect Cost

  Institution

Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715

  Publications

Hernandez, Jose A; Phillips, Aaron H; Erbil, W Kaya et al. (2011) A sterile alpha-motif domain in NafY targets apo-NifDK for iron-molybdenum cofactor delivery via a tethered domain. J Biol Chem 286:6321-8
Martínez-Noël, Giselle; Curatti, Leonardo; Hernandez, Jose A et al. (2011) NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii. Mol Microbiol 79:1182-93
Soboh, Basem; Boyd, Eric S; Zhao, Dehua et al. (2010) Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site. FEBS Lett 584:1487-92
Hernandez, Jose A; Curatti, Leonardo; Aznar, Constantino P et al. (2008) Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor. Proc Natl Acad Sci U S A 105:11679-84
George, Simon J; Igarashi, Robert Y; Piamonteze, Cinthia et al. (2007) Identification of a Mo-Fe-S cluster on NifEN by Mo K-edge extended X-ray absorption fine structure. J Am Chem Soc 129:3060-1
Curatti, Leonardo; Hernandez, Jose A; Igarashi, Robert Y et al. (2007) In vitro synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins. Proc Natl Acad Sci U S A 104:17626-31
Hernandez, Jose A; Igarashi, Robert Y; Soboh, Basem et al. (2007) NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway. Mol Microbiol 63:177-92
Soboh, Basem; Igarashi, Robert Y; Hernandez, Jose A et al. (2006) Purification of a NifEN protein complex that contains bound molybdenum and a FeMo-Co precursor from an Azotobacter vinelandii DeltanifHDK strain. J Biol Chem 281:36701-9
Curatti, Leonardo; Brown, Carolyn S; Ludden, Paul W et al. (2005) Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii. Proc Natl Acad Sci U S A 102:6291-6
Rubio, Luis M; Singer, Steven W; Ludden, Paul W (2004) Purification and characterization of NafY (apodinitrogenase gamma subunit) from Azotobacter vinelandii. J Biol Chem 279:19739-46

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