The reducing activity of glutaredoxin 3 toward cytoplasmic substrate proteins is restricted by methionine 43.
The reducing proteins glutaredoxin 3 (Grx3) and glutaredoxin 1 (Grx1) are structurally similar but exhibit different specificities toward substrates. Grx1 efficiently reduces ribonucleotide reductase and PAPS reductase, while Grx3 reduces these enzymes inefficiently or not at all. We previously described a selection for Grx3 mutants with increased activity toward substrates of Grx1 in vivo. Remarkably, we repeatedly isolated mutants with changes in only one of the amino acids of Grx3, methionine 43, converting it to either valine, leucine, or isoleucine. In this paper we present additional genetic studies and a biochemical characterization of Grx3-Met43Val, the most efficient mutant. We show that Grx3-Met43Val is able to reduce ribonucleotide reductae and PAPS reductase much more efficiently than the wild-type protein in vitro. The altered protein has an increased Vmax over that of Grx3, nearly the same Vmax as Grx1 while the Km remains high. Molecular dynamics simulations suggest that the Met43Val substitution results in changes in properties of the N-terminal cysteine of the active site leading to a considerably lower pKa. Furthermore, Grx3-Met43Val shows an 11 mV lower redox potential than the wild-type Grx3. These findings provide biochemical and structural explanations for the increased reductive efficiency of the mutant Grx3.
Porat
Amir
A
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA.
Lillig
Christopher Horst
CH
Johansson
Catrine
C
Fernandes
Aristi Potamitou
AP
Nilsson
Lennart
L
Holmgren
Arne
A
Beckwith
Jon
J
D000595:Amino Acid Sequence; D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology; D054477:Glutaredoxins; D007700:Kinetics; D008715:Methionine / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
D054477:Glutaredoxins; D008715:Methionine; D010088:Oxidoreductases; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase