Characterization of the Dinuclear Metal Center of Pyrococcus furiosus prolidase and Production of its Mutants with Increased Activity at Low Temperatures

organophosphorus compounds;prolidase;Pyrococcus furiosus

Du, Xuelian ; James Brown, Committee Member,Eric Miller, Committee Member,Robert Upchurch, Committee Member,Shirley Tove, Committee Member,Amy Grunden, Committee Chair,Du, Xuelian ; James Brown ; Committee Member ; Eric Miller ; Committee Member ; Robert Upchurch ; Committee Member ; Shirley Tove ; Committee Member ; Amy Grunden ; Committee Chair

University:North Carolina State University

关键词: organophosphorus compounds;    prolidase;    Pyrococcus furiosus;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/3944/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

Prolidase isolated from the hyperthermophilic archaeon Pyrococcus furiosus has potential for application under harsh conditions for decontamination and detoxification of organophosphorus compounds contained in certain pesticides and chemical warfare agents. However, this application is greatly restricted by two major factors.The first factor comes from current knowledge of this enzyme. Previous study suggested P. furiosus prolidase contained two cobalt atoms with different affinity at the catalytic center which are required for full activation.However, no data has established which Co site was tight-binding and which was loose-binding. To clearly address this question, we used site-directed mutagenesis to modify amino acid residues that participate in binding the Co1 site (E313 and H284), the Co2 site (D209) or bidentate ligand site (E327). Metal-content, enzyme activity and CD-spectra analyses of D209A-, H284L- and E327L-prolidase mutants show that P. furiosus prolidase contains a dinuclear metal center with Co1 serving as the tight-binding and Co2 the loose-binding sites. Results of this study not only provides insight into the nature of P. furiosus prolidase active center, but facilitates our understanding of the mechanisms involved in enzyme catalysis.The second factor that limits the application of P. furiosus prolidase is its narrow temperature range for catalytic activity. P. furiosus prolidase exhibits extreme high activity at 100°C but negligible activity at low temperatures.To improve the enzyme's catalytic activity at low temperatures, P. furiosus prolidases were randomly mutagenized and screened at room temperature for increased activity. This study led to the identification of two low-temperature adapted prolidase mutants with one having Gly39 substituted by glutamate (G39E) and the other having Glu236 substituted by valine (E236V). G39E- and E236V- prolidases were further characterized to obtain better understanding of substrate catalysis at both low and high temperature and the relationship of these features with thermoactivity and thermostability.