Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes.
The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1beta-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1beta(OH)EA versus 3alpha(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1beta(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1beta(OH)EA, 2beta(OH)EA, and 3beta(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted approximately 1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1beta(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1beta(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (kcat) for 1beta(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.
Takahashi
Shunji
S
Plant Physiology, Biochemistry, and Molecular Biology Program, Agronomy Department, University of Kentucky, Lexington, Kentucky, 40546-0312, USA.
Zhao
Yuxin
Y
O'Maille
Paul E
PE
Greenhagen
Bryan T
BT
Noel
Joseph P
JP
Coates
Robert M
RM
Chappell
Joe
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006900:Hydroxylation; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism*; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*
D012717:Sesquiterpenes; D007532:Isoleucine; C081843:capsidiol; D012694:Serine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C436961:cytochrome P-450 CYP71D20 (Nicotiana tabacum)