Title

Structural and functional analysis of metalloproteins in Azotobacter vinelandii

Author

Hanqing Dong

Advisor

Gavini, Nara

Committee Member

Downer, Donald

Committee Member

Wise, Dwayne

Committee Member

Pulakat, Lakshmi

Committee Member

Jung, Yean-sung

Date of Degree

1-1-2007

Original embargo terms

MSU Only Indefinitely

Document Type

Dissertation - Open Access

Major

Biology

Degree Name

Doctor of Philosophy

College

College of Arts and Sciences

Department

Department of Biological Sciences

Abstract

The enzyme nitrogenase, which catalyzes nitrogen fixation in Azotobacter vinelandii, consists of two components, the Fe protein (NifH) and the MoFe protein (NifDK). NifK contains several highly conserved residues implicated in its functions throughout the protein. However, the carboxyl terminus of NifK is not implicated in any of the known functions of this protein. Therefore, the present study explored the role of carboxyl terminal region of NifK. The results of growth analysis showed that when the media was adjusted to be slightly acidic, the strain that expresses the mutated NifK yielded a lower growth compared to the wild type. These observations implied that the carboxyl terminus of the NifK contributes to the formation of a stable nitrogenase complex when A. vinelandii is grown in acidic environment. The proper interaction between NifH and NifK is essential for the nitrogenase conformation. To determine how the interaction is influenced by the characteristics of the amino acids available at position 112 of NifH, we introduced residue mutations to the codon encoding for Glu112. Growth analyses indicated that mutant strains are capable of propagation under nitrogen-deficit conditions although the growth rate is lower than that of wild type strain. Therefore the charge carried by the amino acid at position 112 of NifH plays a minor role in the interaction whereas; a more important factor is the length of the side chains. The research on hydrogenase expressed by bacteria shed light on the possibilities of utilizing this novel energy source. We endeavored to take advantage of the nature of A. vinelandii and construct an A. vinelandii mutant strain expressing Fe-hydrogenase. This ongoing research involves molecular manipulation of the enzyme-encoding gene hydA. The synthetic hydA was incorporated and expressed in A. vinelandii strain DJ54. At the same time, we screened several biomass materials for their capabilities in sustaining diazotrophic growth of A. vinelandii. The result indicated that the HydA protein can be expressed in A. vinelandii under certain conditions and a number of biomass substances can be supportive ingredients for putative biohydrogen media.

Temporal Coverage

2000-2009

URI

https://hdl.handle.net/11668/16262

Comments

hydrogenase||nitrogenase||Azotobacter vinelandii

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