Development of a System for Directed Evolution of Arabidopsis Formate Dehydrogenase to Utilize NADP as a Cofactor

RURALS: Review of Undergraduate Research in Agricultural and Life Sciences

Herman, Patricia L.¹  Markwell, John²  Widhalm, Joshua R.²  Prather, Brittany L.² 

[1] School of Biological Sciences, University of Nebraska-LincolnSchool of Biological Sciences, University of Nebraska-LincolnSchool of Biological Sciences, University of Nebraska-Lincoln;Center for Biological Chemistry, University of Nebraska-LincolnCenter for Biological Chemistry, University of Nebraska-LincolnCenter for Biological Chemistry, University of Nebraska-Lincoln

关键词: Arabidopsis;    formate dehydrogenase;    directed evolution;    NADP;   

DOI  :  

学科分类：农业科学（综合）

来源: University of Nebraska at Lincoln * Institute of Agriculture and Natural Resources

【 摘 要 】

Formate dehydrogenase (FDH) is a NAD-dependent enzyme found in methylotrophic bacteria, yeast and plants. This enzyme catalyzes the reversible oxidation of formate to carbon dioxide. The goal of this research was to determine the feasibility of using a directed evolution approach to generate an altered Arabidopsis FDH with a high affinity for NADP as a cofactor. A PCR procedure that induced approximately 1.5 mutations in the wild-type Arabidopsis FDH sequence per thousand base pairs was developed and the amplified products were transformed into E. coli cells. Approximately 1300 cell lines were assayed in 96-well microplates for activity with NADP+ and 100 putative mutants were selected for further study. One particular mutant line, pFDH-18, possessed reproducible NADP+-FDH activity. Sequence analysis showed that a single T in the wild-type DNA sequence had been changed to a G. The result of this mutation was that an isoleucine (Ile) residue at position 188 in the wild-type enzyme was converted to a methionine. This particular Ile residue is conserved in the known FDH sequences from higher plants and is located in the region of the enzyme that contains the binding domain for the NAD cofactor.

【 授权许可】

Unknown