Role of highly conserved residues in the reaction catalyzed by recombinant Delta7-sterol-C5(6)-desaturase studied by site-directed mutagenesis.
The role of 15 residues in the reaction catalyzed by Arabidopsis thaliana Delta7-sterol-C5(6)-desaturase (5-DES) was investigated using site-directed mutagenesis and expression of the mutated enzymes in an erg3 yeast strain defective in 5-DES. The mutated desaturases were assayed in vivo by sterol analysis and quantification of Delta5,7-sterols. In addition, the activities of the recombinant 5-DESs were examined directly in vitro in the corresponding yeast microsomal preparations. One group of mutants was affected in the eight evolutionarily conserved histidine residues from three histidine-rich motifs. Replacement of these residues by leucine or glutamic acid completely eliminated the desaturase activity both in vivo and in vitro, in contrast to mutations at seven other conserved residues. Thus, mutants H203L, H222L, H222E, P201A, G234A, and G234D had a 5-DES activity reduced to 2-20% of the wild-type enzyme, while mutants K115L, P175V, and P175A had a 5-DES activity and catalytical efficiency (V/K) that was similar to that of the wild-type. Therefore, these residues are not essential for the catalysis but contribute to the activity through conformational or other effects. One possible function for the histidine-rich motifs would be to provide the ligands for a presumed catalytic Fe center, as previously proposed for a number of integral membrane enzymes catalyzing desaturations and hydroxylations [Shanklin et al. (1994) Biochemistry 33, 12787-12794]. Another group of mutants was affected in residue 114 based on previous in vivo observations in A. thaliana indicating that mutant T114I was deficient in 5-DES activity. We show that the enzyme T114I has an 8-fold higher Km and 10-fold reduced catalytic efficiency. Conversely, the functionally conservative substituted mutant enzyme T114S displays a 28-fold higher Vmax value and an 8-fold higher Km value than the wild-type enzyme. Consequently, V/K for T114S was 38-fold higher than that for T114I. The data suggest that Thr 114 is involved in stabilization of the enzyme-substrate complex with a marked discrimination between the ground-state and the transition state of a rate-controlling step in the catalysis by the 5-DES.
Taton
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M
Département d'Enzymologie Moléculaire et Cellulaire, Institut de Biologie Moléculaire des Plantes, CNRS UPR 406, 28 rue Goethe, 67083, Strasbourg Cédex, France.
Husselstein
T
T
Benveniste
P
P
Rahier
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D017343:Genes, Plant; D005822:Genetic Vectors / Q000138:chemical synthesis; D005998:Glycine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011392:Proline / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013912:Threonine / Q000235:genetics
D011994:Recombinant Proteins; C033144:lathosterol delta-5-dehydrogenase; D013912:Threonine; D006639:Histidine; D011392:Proline; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C039306:sterol delta-5 desaturase; D008239:Lysine; D005998:Glycine