This preview shows pages 1–2. Sign up to view the full content.
This preview has intentionally blurred sections. Sign up to view the full version.View Full Document
Unformatted text preview: Eur. J. Biochem. 252 , 458 2 466 ( 1 998) FEBS 1 998 Conferring aldosterone synthesis to human CYP11B1 by replacing key amino acid residues with CYP11B2-specific ones Benjamin BÖTTNER 1 , Karsten DENNER 1 and Rita BERNHARDT 1 ,2 1 Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany 2 Universität des Saarlandes, FR 1 2.4-Biochemie, Saarbrücken, Germany (Received 24 October 1 997/ 1 6 January 1 998) 2 EJB 97 1 5 1 0/4 Performing residue-swapping experiments between the highly conserved human steroidogenic pro- teins CYP 11 B 1 and CYP 11 B2 we recently demonstrated that replacement of specific residues at position 30 1 , 302 and 320 in the aldosterone-producing CYP 11 B2 protein for such residues that were specific for the highly similar cortisol-producing CYP 11 B 1 protein elevated the 11 β-hydroxylase activity dramati- cally. Conversely, aldosterone synthesis in the triple mutant was severely impaired. Here we provide evidence that in a reciprocal experiment, CYP 11 B2-specific amino acids at position 320 and 335 endowed CYP 11 B 1 with an 1 8-oxidase function amounting to 20% of the CYP 11 B2 wild-type activity, thus chang- ing the specificity of steroid hydroxylation by only one point mutation. Combining substitutions at posi- tions 296, 30 1 , 302, 320, 335 and 339 did, however, not result in further enhancement. Paradoxically, 11 β-hydroxylation was not or only marginally affected in CYP 11 B 1 mutants, indicating an alternative structural basis for this activity in CYP 11 B 1 compared with the engineered CYP 11 B2 variant. Our results suggest that the sequence spanned by amino acids 30 1 and 335 constitutes part of the substrate-binding site in CYP 11 B 1 and CYP 11 B2 as well. By constructing chimeric proteins we further investigated the effect of the C-terminal portions of both proteins and found that diverging residues at positions 47 1 , 472, 492, 493 and 494 were insignificant for the stereospecificity and regiospecificity of steroid hydroxylation. Keywords: CYP 11 B 1 ; CYP 11 B2; regiospecificity of steroid hydroxylation; aldosterone synthesis; engi- neering substrate specificity. Glucocorticoids and mineralocorticoids in man are synthe- share a 95% identity [5, 6]. The derived proteins are translated with 503 amino acids and contain an N-terminal mitochondrial sized from cholesterol in different zones of the adrenal cortex. Glucocorticoid biogenesis takes place in the zonae fasciculata/ targeting sequence which after import into the mitochondrial matrix is clipped off yielding the mature proteins of 479 amino reticularis and requires five enzymatic conversions: cleavage of the cholesterol side chain producing pregnenolone, 1 7 A-hydrox- acids. The latter function in context with two accessory proteins: adrenodoxin reductase, a flavoprotein [7, 8] and adrenodoxin, a ylation and 3 β-dehydrogenation to yield 1 7-hydroxyprogester- one and successive hydroxylation steps at the 2 1 and 11 β posi- soluble iron-sulfur protein  transporting...
View Full Document
- Spring '10