Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein.
Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys(60) and Gly(61) are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly(115) and Gly(116) could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys(117). Substitution of Phe(50) alters the beta-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.
Bellí
Gemma
G
Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Rovira Roure 44, 25198-Lleida, Spain.
Polaina
Julio
J
Tamarit
Jordi
J
De La Torre
María Angeles
MA
Rodríguez-Manzaneque
María Teresa
MT
Ros
Joaquim
J
Herrero
Enrique
E
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D054477:Glutaredoxins; D006207:Half-Life; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010088:Oxidoreductases; D010957:Plasmids; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D005656:Fungal Proteins; D054477:Glutaredoxins; C516012:Grx4 protein, S cerevisiae; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D007501:Iron; D010088:Oxidoreductases