Expression of human CYP27B1 in Escherichia coli and characterization in phospholipid vesicles

Abstract

CYP27B1 is a mitochondrial cytochrome P450 that catalyses the hydroxylation of 25-hydroxyvitamin D3 at the C1α-position to give the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3. We successfully expressed human CYP27B1 in Escherichia coli and partially purified this labile enzyme and carried out a detailed characterization of its kinetic properties in a reconstituted membrane environment. The phospholipid concentration did not affect the enzyme activity in the vesicle-reconstituted system, although it was influenced by the phospholipid composition, with the addition of cardiolipin lowering the Km for 25-hydroxyvitamin D3. These data are consistent with the enzyme accessing substrate from the hydrophobic domain of the vesicle membrane. Cardiolipin also caused the appearance of inhibition of activity at high substrate concentrations. This substrate inhibition fitted a model for one catalytic and two inhibitory sites on the enzyme for the binding of substrate. The Km for human adrenodoxin was observed to decrease with decreasing substrate concentration, with the catalytic efficiency (kcat/K m) being largely independent of adrenodoxin concentration. Human CYP27B1 was also active on 25-hydroxyvitamin D2 and on intermediates of the CYP24A1-mediated inactivation pathway, 24R,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, with all these substrates showing comparable kcat values of 50-71 min-1, similar to 25-hydroxyvitamin D3. The latter two substrates gave higher K m values than that for 25-hydroxy-vitamin D3. The present study shows that human CYP27B1 can be partially purified in an active form with the enzyme displaying high activity towards a range of substrates in a phospholipid vesicle-reconstituted system that mimics the inner-mitochondrial membrane. Human CYP27B1 was expressed in E. coli, partially purified and characterised in a reconstituted phospholipid membrane. Parameters examined included adrenodoxin concentration, phospholipid composition and secosteroid side-chain structure. We showed that human CYP27B1 is active towards a range of substrates including 25-hydroxyvitamins D3 and D2, and 24R,25-dihydroxyvitamin D3 in phospholipid vesicles, with some substrates causing inhibition at high concentrations. © 2012 The Authors Journal compilation © 2012 FEBS.