title: Roles of four conserved basic amino acids in a ferredoxin-dependent cyanobacterial nitrate reductase.
abstract: The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, United States.
authors.forename: Anurag P
authors.initials: AP
authors.lastname: Srivastava
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hirasawa
authors.forename: Megha
authors.initials: M
authors.lastname: Bhalla
authors.forename: Jung-Sung
authors.initials: JS
authors.lastname: Chung
authors.forename: James P
authors.initials: JP
authors.lastname: Allen
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
authors.forename: Jatindra N
authors.initials: JN
authors.lastname: Tripathy
authors.forename: Luis M
authors.initials: LM
authors.lastname: Rubio
authors.forename: Brian
authors.initials: B
authors.lastname: Vaccaro
authors.forename: Sowmya
authors.initials: S
authors.lastname: Subramanian
authors.forename: Enrique
authors.initials: E
authors.lastname: Flores
authors.forename: Masoud
authors.initials: M
authors.lastname: Zabet-Moghaddam
authors.forename: Kyle
authors.initials: K
authors.lastname: Stitle
authors.forename: David B
authors.initials: DB
authors.lastname: Knaff
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D024361:Amino Acids, Basic / Q000737:chemistry*; D017124:Conserved Sequence; D005288:Ferredoxins / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D050901:Nitrate Reductase / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D046940:Synechococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D024361:Amino Acids, Basic; D005288:Ferredoxins; D005973:Glutamine; D050901:Nitrate Reductase