Cytochrome P450 125 (CYP125) catalyses C26-hydroxylation to initiate sterol side-chain degradation in Rhodococcus jostii RHA1

Abstract

The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3-hydroxysterols such as cholesterol, but not on 3-oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side-chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side-chain. The RG32 Omega cyp125 mutant failed to transform the side-chain of cholesterol, but degraded that of 5-cholestene-26-oic acid-3beta-ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125(RHA1) was < 10% in the low-spin state, cholesterol (K(D)(app) = 0.20 +/- 0.08 microM), 5 alpha-cholestanol (K(D)(app) = 0.15 +/- 0.03 microM) and 4-cholestene-3-one (K(D)(app) = 0.20 +/- 0.03 microM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26-carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.

Fig. 1. The initial steps of aerobic cholesterol degradation in bacteria (Sih et al., 1968a,b; Szentirmai, 1990; van der Geize et al., 2007)

A. Steroid nomenclature. B. CYP125 is involved in steroid C26 hydroxylation. Subsequent oxidation leads to a C26-oic acid metabolite. C. Sterol degradation proceeds via steroid ring oxidation and side-chain degradation (upper route). The exact order of side-chain degradation and ring oxidation in vivo is unknown and may vary between microorganisms. In R. jostii RHA1, ring oxidation is not initiated until sometime after the side-chain attack by CYP125 (dotted arrow). The depicted metabolites are: (I) 5-cholestene-3β-ol (cholesterol), (II) 4-cholestene-3-one, (III) 5-cholestene-26-oic acid-3β-ol, (IV) 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ^1,4-BNC), (V) 1,4-androstadiene-3,17-dione and (VI) 2-hydroxyhexa-2,4-diene-oic acid. R. rhodochrous mutant strain RG32 (see text) converts compound I into compounds IV and V by selective side-chain degradation. Abbreviations: CYP125, steroid 26-monooxygenase; CHO, cholesterol oxidase; 3βHSD, 3β-hydroxysteroid dehydrogenase (Yang et al., 2007).

Fig. 2

Cholesterol degradation by cell cultures of strains RHA1 and RHA1Δcyp125 grown in mineral liquid media supplemented with pyruvate (20 mM) and cholesterol (2.5 mM). The data represent averages of triplicates. Error bars indicate standard deviations.

Fig. 3

HPLC profiles of whole-cell biotransformations of cholesterol by cell cultures of (A) R. rhodochrous strain RG32 showing the formation of 1,4-androstadiene-3,17-dione (ADD) and 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ^1,4-BNC) via selective sterol side-chain degradation, (B) mutant strain RG32Ωcyp125 and (C) cyp125_DSM43269 complemented mutant strain RG32Ωcyp125. HPLC profiles of whole-cell biotransformations of 5-cholenic acid-3β-ol (D) and 5-cholestene-26-oic acid-3β-ol (E) by cell cultures of R. rhodochrous mutant strain RG32Ωcyp125 are also shown. Profiles of authentic ADD (50 μM, F) and Δ^1,4-BNC (G), obtained by incubating authentic 3-oxo-23,24-bisnorchol-4-en-22-oic acid (50 μM, Δ⁴-BNC) with purified Δ1-KSTD1 (Knol et al., 2008), are included as reference samples. Insets: GC profiles showing the accumulation of 4-cholestene-3-one (2), 1,4-cholestadiene-3-one (3) and 5α-cholestane-3-one (4) from cholesterol (1) by R. rhodochrous mutant strain RG32Ωcyp125, but not strain RG32.

Fig. 4

The absorption spectrum of CYP125_RHA1 in the oxidized state as isolated (solid line) and incubated with 10 μM cholesterol in oxidized (dashed line) and reduced (dotted line) states. The inset shows the reduced CO-difference spectrum of the enzyme incubated with 10 μM cholesterol. The sample contained 2.9 μM purified CYP125_RHA1, 0.1 M potassium phosphate buffer, pH 7.0, 25°C; cholesterol was added from a 1 mM stock solubilized in 10% 2-hydroxypropyl-β-cyclodextrin.

Fig. 5. Binding of steroids to purified CYP125_RHA1

A. Spectral responses of 3.7 μM purified CYP125_RHA1 induced by 10 μM cholesterol (solid line) and 10 μM 5-cholestene-26-oic acid-3β-ol (dashed line). The dependence of the absorbance change of CYP125_RHA1 at 422 nm on (B) cholesterol, (C) 5α-cholestanol and (D) 4-cholestene-3-one concentration. The best fit of Eq. 1 to the data as determined using R is represented as a grey line with fitted parameters K_D = 0.20 ± 0.08 μM, ΔA_max = 0.0298 ± 0.0006, and [E] = 4.0 ± 0.2 μM for cholesterol; K_D = 0.15 ± 0.03 μM, ΔA_max = 0.0293 ± 0.0002, and [E] = 4.3 ± 0.1 μM for 5α-cholestanol; and K_D = 0.20 ± 0.03 μM, ΔA_max = 0.0300 ± 0.0002, and [E] = 3.6 ± 0.1 μM for 4-cholestene-3-one. Steroids were prepared as stock solutions in 10% 2-hydroxypropyl-β-cyclodextrin which alone did not induce a CYP125_RHA1 spectral response.