Structural and Functional Relationships of Laurate (omega-1)-Hydroxylase (P-450)
| Project/Area Number |
63580153
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
代謝生物化学
|
|---|
| Research Institution | University of Osaka Prefecture (1989)
Osaka University (1988) |
|---|
Principal Investigator |
IMAI Yoshio University of Osaka Prefecture, College of Agriculture, Professor, 農学部, 教授 (60029949)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
IMAI Yoshio University of Osaka Prefecture, College of Agriculture, Professor (60029949)
|
|---|
| Project Period (FY) |
1988 – 1989
|
| Project Status |
Completed (Fiscal Year 1989)
|
| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,400,000 (Direct Cost: ¥1,400,000)
|
|---|
| Keywords | P-450 / Laurate Hydroxylase / Testosterone Hydroxylase / Chimeric Enzyme / Site-Directed Mutagenesis / Substrate Specificity / Structure-Function Relationships / Expression of Animal Protein in Yeast Cells / P-450 / チトクロムP450 / キメラ酵素 / CDNAの点変異 / 人工的変異酵素 / 酵素の機能部位 / 一次構造と基質特異性 |
|---|
| Research Abstract |
cDNAs for chimeras of rabbit liver P-450s (laurate (omega-1)-hydroxylase and testosterone 16 alpha-hydrox- ylase) and site-specific mutants of the laurate hydroxylase were constructed, and expressed in yeast cells utilizing DNA manipulation techniques. The P-450s thus syntesized were purified, and their propenires were examined to identify domains of laurate (omega-1)-hydroxylase responsible for its substrate specificity.
1. Two segments of laurate (omega-1)-hydroxylase covering residues 90-125 and 210-252 constitute the substrate binding domain and cooperate to fix the substrate on the hydroxylase molecule. The chimeras containing the sequences of the laurate hydroxylase in the both regions are active in the hydroxylation of the fatty acids, while the chimeras devoid of these sequences in either of the two regions were practically inactive and exhibited lower affinity for the fatty acids than the wild-type hydroxylase. The chimeras containing neither of these sequences could not bind t
… More
he substrate.
2. When the the carboxy-terminal 28 residues of the laurate hydroxylase were replaced by the corresponding sequence of testosterone 16 alpha-hydroxylase, the laurate hydroxylase activity increased about 2.5 times and, moreover, a new stereospecific hydroxylase activity (16 beta-hydroxylation of testosterone) appeared. These observations suggest that the laurate hydroxylase contains a structure that is capable of binding testosterone at a proper orientation for the hydroxylation of the steroid at the beta position, but the carboxy-terminal segment of the laurate hydroxylase prevents the testosterone molecule from gaining access to the binding site while that of the testosterone hydroxylase does not.
3. Replacement of Thr-301 and/or Thr-302 from the laurate hydroxylase by other amino acid residues affected the rates of the laurate and caprate hydroxylations at the omega-1 and the omega positions, indicating that these residues play an important role in recognizing the difference in the chain length between the two fatty acids and determining the position in the substrates to be hydroxylated. Less
|
Report
(3 results)
Research Products
(23 results)
