Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin.
A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed Zm8,7SI, has been isolated from an EST (expressed sequence tag) library and subcloned into the yeast erg2 mutant lacking 8,7SI activity. Zm8,7SI restored endogenous ergosterol synthesis. An in vitro enzymatic assay in the corresponding yeast microsomal extract indicated that the preferred Delta(8)-sterol substrate possesses a single C4alpha methyl group, in contrast with 8,7SIs from animals and fungi, thus reflecting the diversity in the structure of their active site in relation to the distinct sterol biosynthetic pathways. In accordance with the proposed catalytic mechanism, a series of lipophilic ammonium-ion-containing derivatives possessing a variety of structures and biological properties, potently inhibited the Zm8,7SI in vitro. To evaluate the importance of a series of conserved acidic and tryptophan residues which could be involved in the Zm8,7SI catalytic mechanism, 20 mutants of Zm8,7SI were constructed as well as a number of corresponding mutants of the Saccharomyces cerevisiae 8,7SI. The mutated isomerases were assayed in vivo by sterol analysis and quantification of Delta(5,7)-sterols and directly in vitro by examination of the activities of the recombinant Zm8,7SI mutants. These studies have identified His(74), Glu(78), Asp(107), Glu(121), Trp(66) and Trp(193) that are required for Zm8,7SI activity and show that binding of the enzyme-substrate complex is impaired in the mutant T124I. They underline the functional homology between the plant and animal 8,7SIs on one hand, in contrast with the yeast 8,7SI on the other hand, in accordance with their molecular diversity and distinct mechanisms.
Rahier
Alain
A
Institut de Biologie Moléculaire des Plantes, CNRS, UPR2357, 28 rue Goethe, 67083 Strasbourg cedex, France. alain.rahier@ibmp-ulp.u-strasbg.fr
Pierre
Sylvain
S
Riveill
Geneviève
G
Karst
Francis
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D005658:Fungi / Q000201:enzymology* / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D014644:Genetic Variation; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013251:Steroid Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013261:Sterols / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D044366:Transition Temperature; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D010940:Plant Proteins; D011994:Recombinant Proteins; D013261:Sterols; D013251:Steroid Isomerases; C022669:delta(8)-delta(7)-sterol isomerase