Crystal Structure of CYP105A1 (P450SU-1) in Complex with 1α,25-Dihydroxyvitamin D₃^,

Abstract

Vitamin D 3 (VD 3), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD 3 requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD 3 into 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1alpha,25(OH) 2D 3. The compound 1alpha,25(OH) 2D 3 is positioned 11 A from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1-VD 3 complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B' and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1alpha-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD 3.

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

“…Comparison with the CYP105AS1 open structure shows that compactin binding to P450 Prava reorientates the BC-loop region (with the Pro82-Ala88 region now disordered) and reorganizes the FG helices. This closing motion shields the ligand and active site from bulk solvent, as previously observed in other CYP105 enzymes (26,27). Compactin displaces the aqua-sixth ligand from the heme iron and is bound in an active conformation with the C6 carbon placed within 4.7 Å of the heme iron (Fig.…”

“…The CYP105AS1 structure was solved to 2.05 Å using molecular replacement with the vitamin D hydroxylase CYP105A1 (26) [Protein Data Bank (PDB) ID code 2ZBY; 44% sequence identity]. The final structure contains residues Glu10 to Asp400 with the exception of the polypeptide stretch from Ser77 to Ala85, with an RMSD of 1.38 Å for 355 C a atoms (Fig.…”

“…Data were reduced/scaled with the XDS suite (47) and processed using the CCP4 suite (48). The WT CYP105AS1 structure was solved using molecular replacement with the CYP105A1 structure as a search model (PDB ID code 2ZBY) (26), and the P450 Prava mutant was solved using the WT CYP105AS1 structure. Structures were refined using Refmac5 (48) and COOT (49).…”

Diversity and Substrate Specificity in the Structures of Steroidogenic Cytochrome P450 Enzymes

“…36,37) Comparison of the main chain folds between CYP2R1 and CYP105A1 shows a large difference in the F-G loop region where CYP105A1 lacks extra helices F' and G', as do other bacterial CYPs.…”

Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis : Conformational Flexibility and Histidine Ligation State

“…P450nor, one of the most unusual P450 enzymes, catalyzes the reduction of nitric oxide to dinitrogen oxide in the fungal denitrification steps, utilizing NADH as the direct electron donor (27,31). To date, the crystal structures of two CYP105 family enzymes, P450 MoxA and P450 SU-1 (CYP105A1), have been reported (49,54). Although their biological roles remain unclear, the ability of these enzymes to hydroxylate a wide variety of compounds attracts considerable interest in various applications.…”