Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3β-hydroxysteroid dehydrogenase/Isomerase1

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Abstract

3β-Hydroxysteroid dehydrogenase/steroid Δ5→4-isomerase (3β-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants – H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3β-HSD/isomerase (monomeric Mr=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.0 l). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3β-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3β-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3β-HSD activity. The 3β-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3β-HSD activity, and Tyr253 or Tyr254 participates in the isomerase activity of human type I (placental) enzyme.

Introduction

Human placental 3β-hydroxysteroid dehydrogenase (3β-HSD, EC 1.1.1.145) and steroid Δ5→4-isomerase (EC 5.3.3.1) catalyze the sequential conversion of 3β-hydroxy-5-ene steroids (pregnenolone, dehydroepiandrosterone) to 3-oxo-4-ene steroids (progesterone, androstenedione) on a single, dimeric protein containing both enzyme activities[1]. The bifunctional enzyme catalyzes a key biosynthetic step that is required for each of the pathways that produce active steroid hormones. In the human, the type I enzyme (placenta, skin, mammary gland) and the type II enzyme (gonads, adrenals) are expressed in a tissue-specific pattern, are encoded by two distinct genes, and have 93% homology in the amino acid sequences[2]. The 3β-HSD and isomerase activities co-purify as the single type I protein from human term placenta1, 3.

Tryptic peptides associated with both catalytic activities have been localized using affinity radiolabeling steroids. One tryptic peptide, 250GQFYYISDDTPHQSYDNLNYTLSK273, was modified at His261 by both the 3β-HSD-site-directed alkylator, 2α-bromoacetoxyprogesterone4, 5and by the isomerase-site-directed alkylator, 5,10-secoestr-4-yne-3,10,17-trione[6]. Additional studies using stopped-flow fluorescent spectroscopy[7]supported the following model for the sequential reaction mechanism: NADH, the coenzyme product of the rate-limiting 3β-HSD reaction, induces a conformational change around the bound 3-oxo-5-ene-steroid (the 3β-HSD product and isomerase substrate) to activate isomerase in the single, bifunctional enzyme protein. Thus, 3β-HSD/isomerase offers a unique enzyme system to study using site-directed mutagenesis because an appropriately targeted point mutation may modify only one of the two activities. In the current study, we have used site-directed mutagenesis to produce three mutated forms of human type I (placental) 3β-HSD/isomerase (H261R, Y253F and Y253,254F) and compared the kinetics of the purified mutant, wild-type, and native placental enzymes to investigate the roles of the targeted amino acid residues in the 3β-HSD and isomerase reaction mechanisms.

Section snippets

Materials

Pregnenolone, dehydroepiandrosterone and pyridine nucleotides were purchased from Sigma (St. Louis, MO); 5-androstene-3,17-dione and 5-pregnene-3,20-dione from Steraloids (Wilton, NH); reagent grade salts, chemicals and analytical grade solvents from Fisher (Pittsburg, PA). Glass distilled, deionized water was used for all aqueous solutions.

Site-directed mutagenesis

The 1,555 base pair (bp) cDNA that encodes human type I (placental) 3β-HSD/isomerase was cut from the host plasmid, pCMV5H3βHSD[8], using EcoR1/PspA1 and

Expression of the mutant and wild-type enzymes

A Western blot of an SDS-polyacrylamide gel (Fig. 1) showed that Sf9 cells infected with the appropriate recombinant baculovirus expressed the immunoreactive H261R (lane 2), Y253,254F (lane 3), Y253F (lane 4) and wild type (lane 6) protein, all of which co-migrated with the purified, human placental 3β-HSD/isomerase (lane 1). The absence of a band in lane 5 shows that uninfected Sf9 cells do not express endogenous 3β-HSD/isomerase.

Kinetic analysis of the purified mutants

Table 1 summarizes the results of the substrate kinetic studies

Discussion

The 3β-HSD/isomerase enzymes present an interesting mechanistic question: Are the catalytic centers and critical amino acids for each of the two coupled, biosynthetic reactions located in unique and separate domains, in overlapping but distinct sites, or in the same, inseparable site? We have identified the active site region for 3β-HSD and isomerase in the primary structure of the enzyme using a substrate-protection technique that enhances the specificity of affinity radiolabeling steroids4, 5

References (22)

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1

Supported by NIH Grant HD20055 (JLT).

2

Present address: Department of Obstetrics and Gynecology, Henry Ford Health System, Area K-9, 2799 West Grand Boulevard, Detroit, MI 48202, U.S.A.

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