Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization.
The human emopamil binding protein (hEBP) exhibits sterol Delta8-Delta7 isomerase activity (EC 5.3.3.5) upon heterologous expression in a sterol Delta8-Delta7 isomerization-deficient erg2-3 yeast strain. Ala scanning mutagenesis was used to identify residues in the four putative transmembrane alpha-helices of hEBP that are required for catalytic activity. Isomerization was assayed in vivo by spectrophotometric quantification of Delta5,7-sterols. Out of 64 Ala mutants of hEBP only H77A-, E81A-, E123A-, T126A-, N194A-, and W197A-expressing yeast strains contained 10% or less of wild-type (wt) Delta5,7-sterols. All substitutions of these six residues with functionally or structurally similar amino acid residues failed to fully restore catalytic activity. Mutants E81D, T126S, N194Q, and W197F, but not H77N and E123D, still bound the enzyme inhibitor 3H-ifenprodil. Changed equilibrium and kinetic binding properties of the mutant enzymes confirmed our previous suggestion that residues required for catalytic activity are also involved in inhibitor binding [Moebius et al. (1996) Biochemistry 35, 16871-16878]. His77, Glu81, Glu123, Thr126, Asn194, and Trp197 are localized in the cytoplasmic halves of the transmembrane segments 2-4 and are proposed to line the catalytic cleft. Ala mutants of Trp102, Tyr105, Asp109, Arg111, and Tyr112 in a conserved cytoplasmic domain (WKEYXKGDSRY) between transmembrane segments 2 and 3 contained less than 10% of wt Delta5,7-sterols, implying that this region also could be functionally important. The in vivo complementation of enzyme-deficient yeast strains with mutated cDNAs is a simple and sensitive method to rapidly analyze the functional consequences of mutations in sterol modifying enzymes.
Moebius
F F
FF
Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
Soellner
K E
KE
Fiechtner
B
B
Huck
C W
CW
Bonn
G
G
Glossmann
H
H
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013251:Steroid Isomerases / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D010880:Piperidines; C033144:lathosterol delta-5-dehydrogenase; D013912:Threonine; D018698:Glutamic Acid; D006639:Histidine; D001216:Asparagine; D014364:Tryptophan; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C039306:sterol delta-5 desaturase; D013251:Steroid Isomerases; C022669:delta(8)-delta(7)-sterol isomerase; C090445:EBP protein, human; D000409:Alanine; C010739:ifenprodil