CYP106A2-A versatile biocatalyst with high potential for biotechnological production of selectively hydroxylated steroid and terpenoid compounds.

CYP106A2 from Bacillus megaterium ATCC13368, was identified in the 1970s as one of the first bacterial steroid hydroxylases responsible for the conversion of progesterone to 15ß-hydroxyprogesterone. Later on it has been proven to be a potent hydroxylase of numerous 3-oxo-Δ4 as well as 3-hydroxy-Δ5-steroids and has recently also been characterized as a regioselective allylic bacterial diterpene hydroxylase. The main hydroxylation position of CYP106A2 is thought to be influenced by the functional groups at C3 position in the steroid core leading to a favored 15ß-hydroxylation of 3-oxo-Δ4-steroids and 7ß-hydroxylation of 3-hydroxy-Δ5-steroids. However, in some cases the hydroxylation is not strictly selective, resulting in the formation of undesired side-products. To overcome the unspecific hydroxylations or, on the contrary, to gain more of these products in case they are of industrial interest, rational protein design and directed evolution have been successfully performed to shift the stereoselectivity of hydroxylation by CYP106A2. The subsequently obtained hydroxylated steroid and terpene derivatives are especially useful as drug metabolites and drug precursors for the pharmaceutical industry, due to their diverse biological properties and hardship of their chemical synthesis. As a soluble prokaryotic P450 with broad substrate spectrum and hydroxylating capacity, CYP106A2 is an outstanding candidate to establish bioconversion processes. It has been expressed with respectable yields in Escherichia coli and Bacillus megaterium and was applied for the preparative hydroxylation of several steroids and terpenes. Recently, the application of the enzyme was assessed under process conditions as well, depicting a successfully optimized process development and getting us closer to industrial scale process requirements and a future large scale application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Crystal Structure of CYP106A2 in Substrate-Free and Substrate-Bound Form.

CYP106A2 from Bacillus megaterium ATCC 13368 is known as a bacterial steroid hydroxylase that is also capable of hydroxylating a variety of terpenoids. To analyze the substrate specificity of this enzyme further, different resin acids of the abietane and pimarane types were tested with regard to binding and conversion. Product formation could be shown for all tested substrates. Spectroscopic studies revealed type I binding spectra for isopimaric acid, but dehydroabietic acid did not induce a high-spin shift of the enzyme. Interestingly, binding of abietic acid resulted in a type II difference spectrum typical for nitrogenous inhibitors. Co-crystallization of CYP106A2 with abietic acid and structure determination revealed bending of the heme cofactor when abietic acid was bound in the active site. Quantum chemical calculations strongly suggest that this heme distortion is the cause of the unusual spectroscopic characteristics.

Terpene hydroxylation with microbial cytochrome P450 monooxygenases.

Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes.

Structural and functional analysis of novel human cytochrome C targets in apoptosis.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.

New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants.

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.

Design and characterization of an efficient CYP105A1-based whole-cell biocatalyst for the conversion of resin acid diterpenoids in permeabilized Escherichia coli.

Cytochrome P450 enzymes exhibit a tremendous potential for biotechnological applications due to their ability to introduce oxygen into non-activated carbon atoms. Their catalytic diversity is complemented by a broad substrate range covering many natural compounds. Especially the functionalization of terpenoids by P450s becomes increasingly interesting due to the diverse biological effects of these compounds. The bacterial CYP105A1 from Streptomyces griseolus was recently identified to carry out a one-step hydroxylation of several abietane-type resin acids. In this work, a whole-cell system for CYP105A1 with its heterologous electron transfer proteins Arh1 and Etp1(fd) from Schizosaccharomyces pombe was designed in Escherichia coli JM109 cells. Additionally, an enzyme-coupled cofactor regeneration system was integrated by co-expression of alcohol dehydrogenase from Lactobacillus brevis. In order to overcome mass transfer limitations of substrate into the cell, different agents were tested towards their permeabilizing activity on the E. coli membrane. The peptide antibiotic polymyxin B proved to be the most effective permeabilizer. After optimising the expression and conversion conditions, the cells were able to completely convert 200 µM of abietic acid into 15-hydroxyabietic acid within 2 h, exhibiting an initial conversion rate of 125 µM/h. These results demonstrate the high potential of this whole-cell system for the synthesis of functionalized resin acid diterpenoids.

Novel family members of CYP109 from Sorangium cellulosum So ce56 exhibit characteristic biochemical and biophysical properties.

The members of the CYP109 family (CYP109C1, CYP109C2, and CYP109D1) from Sorangium cellulosum So ce56 are among the 21 P450 enzymes, of which only CYP109D1 and CYP264B1 have so far been functionally characterized. Here, we attempted to characterize two other P450s (CYP109C1 and CYP109C2) for the first time and compare their biochemical, biophysical, and functional properties to those of the fatty acid hydroxylating CYP109D1. Considering the physiological importance of fatty acids, we investigated saturated fatty acid binding and conversion for all members of the CYP109 family. The interaction between the CYP109 members and different autologous/heterologous redox partners was compared using Biacore measurements in which only CYP109D1 and bovine adrenodoxin (Adx) formed a complex. Surprisingly, this interaction was similarly efficient as the interaction of Adx with its mammalian redox partners. The in vitro reconstitution assays showed no activity when using CYP109C1, although substrate binding was demonstrated; also, there was subterminal hydroxylation of saturated fatty acids, when using CYP109C2 and CYP109D1, where CYP109D1 was a much more efficient fatty acid hydroxylase. Interestingly, the hydroxylation position moved inside the fatty acid chain when using long-chain fatty acids, thus producing possible precursors for physiologically important products.

Resin acid conversion with CYP105A1: an enzyme with potential for the production of pharmaceutically relevant diterpenoids.

Cytochrome P450s are very versatile enzymes with great potential for biotechnological applications because of their ability to oxidize unactivated CH bonds. CYP105A1 from Streptomyces griseolus was first described as a herbicide-inducible sulfonylurea hydroxylase, but it is also able to convert other substrates such as vitamin D(3) . To extend the substrate pool of this interesting enzyme further, we screened a small diterpenoid compound library and were able to show the conversion of several resin acids. Binding of abietic acid, dehydroabietic acid, and isopimaric acid to the active site was assayed, and V(max) and K(m) values were calculated. The products were analyzed by NMR spectroscopy and identified as 15-hydroxyabietic acid, 15-hydroxydehydroabietic acid, and 15,16-epoxyisopimaric acid. As the observed products are difficult to obtain by chemical synthesis, CYP105A1 has proved to be a promising candidate for biotechnological applications that combine bioconversion and chemical synthesis to obtain functionalized resin acids.

Substitution of lysine with glutamic acid at position 193 in bovine CYP11A1 significantly affects protein oligomerization and solubility but not enzymatic activity.

CYP11A1, a mitochondrial cytochrome P450, catalyzes the conversion from cholesterol to pregnenolone, the crucial step in the steroid hormone biosynthesis of mammals. It was shown in prior investigations, that the putative F-G loop of this enzyme is involved in membrane attachment. We produced different bovine CYP11A1 variants by rational protein design and could show that a deletion of 20 amino acids comprising parts of the F-G loop results in an enzyme with a three-fold increased solubility, the highest solubility of a CYP11A1 variant obtained so far. Furthermore, a single amino acid mutation, K193E, could be identified which leads not only to a higher solubility of CYP11A1 as well as a 4-fold improved expression rate, but also lowers the oligomerization of the protein while its activity is only slightly decreased. Therefore, this mutant has many advantages for the biotechnological application of CYP11A1 and is an important step towards crystallization of this mitochondrial P450.