Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium.

Cytochrome P450 monooxygenases (P450s) are attractive enzymes for the pharmaceutical industry, in particular, for applications in steroidal drug synthesis. Here, we report a comprehensive functional and structural characterization of CYP109E1, a novel steroid-converting cytochrome P450 enzyme identified from the genome of Bacillus megaterium DSM319. In vitro and whole-cell in vivo turnover experiments, combined with binding assays, revealed that CYP109E1 is able to hydroxylate testosterone at position 16ß. Related steroids with bulky substituents at carbon C17, like corticosterone, bind to the enzyme without being converted. High-resolution X-ray structures were solved of a steroid-free form of CYP109E1 and of complexes with testosterone and corticosterone. The structural analysis revealed a highly dynamic active site at the distal side of the heme, which is wide open in the absence of steroids, can bind four ordered corticosterone molecules simultaneously, and undergoes substantial narrowing upon binding of single steroid molecules. In the crystal structures, the single bound steroids adopt unproductive binding modes coordinating the heme-iron with their C3-keto oxygen. Molecular dynamics (MD) simulations suggest that the steroids may also bind in ~180° reversed orientations with the C16 carbon and C17-substituents pointing toward the heme, leading to productive binding of testosterone explaining the observed regio- and stereoselectivity. The X-ray structures and MD simulations further identify several residues with important roles in steroid binding and conversion, which could be confirmed by site-directed mutagenesis. Taken together, our results provide unique insights into the CYP109E1 activity, substrate specificity, and regio/stereoselectivity. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5L90 (steroid-free CYP109E1), 5L91 (CYP109E1-COR4), 5L94 (CYP109E1-TES), and 5L92 (CYP109E1-COR). ENZYMES: Cytochrome P450 monooxygenase CYP109E1, EC 1.14.14.1, UniProt ID: D5DKI8, Adrenodoxin reductase EC 1.18.1.6.

Semirational Protein Engineering of CYP153AM.aq. -CPRBM3 for Efficient Terminal Hydroxylation of Short- to Long-Chain Fatty Acids.

The regioselective terminal hydroxylation of alkanes and fatty acids is of great interest in a variety of industrial applications, such as in cosmetics, in fine chemicals, and in the fragrance industry. The chemically challenging activation and oxidation of non-activated C-H bonds can be achieved with cytochrome P450 enzymes. CYP153AM.aq. -CPRBM3 is an artificial fusion construct consisting of the heme domain from Marinobacter aquaeolei and the reductase domain of CYP102A1 from Bacillus megaterium. It has the ability to hydroxylate medium- and long-chain fatty acids selectively at their terminal positions. However, the activity of this interesting P450 construct needs to be improved for applications in industrial processes. For this purpose, the design of mutant libraries including two consecutive steps of mutagenesis is demonstrated. Targeted positions and residues chosen for substitution were based on semi-rational protein design after creation of a homology model of the heme domain of CYP153AM.aq. , sequence alignments, and docking studies. Site-directed mutagenesis was the preferred method employed to address positions within the binding pocket, whereas diversity was created with the aid of a degenerate codon for amino acids located at the substrate entrance channel. Combining the successful variants led to the identification of a double variant-G307A/S233G-that showed alterations of one position within the binding pocket and one position located in the substrate access channel. This double variant showed twofold increased activity relative to the wild type for the terminal hydroxylation of medium-chain-length fatty acids. This variant furthermore showed improved activity towards short- and long-chain fatty acids and enhanced stability in the presence of higher concentrations of fatty acids.

Enantioselective Benzylic Hydroxylation Catalysed by P450 Monooxygenases: Characterisation of a P450cam Mutant Library and Molecular Modelling.

Cytochrome P450 monooxygenases can catalyse the stereoselective C-H activation of a very broad range of substrates. Prediction and control of enantioselectivity of this enzyme class is of great interest for the synthesis of high-value chiral molecules. Here we have used a combination of molecular dynamics simulations and experimental screening to study the enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene. Small variations either in substrate structure or in enzyme active site architecture were shown to lead to dramatic changes in enantioselectivity; this was broadly in agreement with computational predictions. In addition to validating computational approaches, these studies have provided us with a deeper understanding of effects that might control stereoselectivity in these biooxidation reactions.

Dendritic targeting of short and long 3' UTR BDNF mRNA is regulated by BDNF or NT-3 and distinct sets of RNA-binding proteins.

Sorting of mRNAs in neuronal dendrites relies upon inducible transport mechanisms whose molecular bases are poorly understood. We investigated here the mechanism of inducible dendritic targeting of rat brain-derived neurotrophic factor (BDNF) mRNAs as a paradigmatic example. BDNF encodes multiple mRNAs with either short or long 3' UTR, both hypothesized to harbor inducible dendritic targeting signals. However, the mechanisms of sorting of the two 3' UTR isoforms are controversial. We found that dendritic localization of BDNF mRNAs with short 3' UTR was induced by depolarization and NT3 in vitro or by seizures in vivo and required CPEB-1, -2 and ELAV-2, -4. Dendritic targeting of long 3' UTR was induced by activity or BDNF and required CPEB-1 and the relief of soma-retention signals mediated by ELAV-1, -3, -4, and FXR proteins. Thus, long and short 3' UTRs, by using different sets of RNA-binding proteins provide a mechanism of selective targeting in response to different stimuli which may underlay distinct roles of BDNF variants in neuronal development and plasticity.

Identification of universal selectivity-determining positions in cytochrome P450 monooxygenases by systematic sequence-based literature mining.

Cytochrome P450 monooxygenases (CYPs) are a large, highly diverse protein family with a common fold. The sequences, structures, and functions of CYPs have been extensively studied resulting in more than 53,000 scientific articles. A sequence-based literature mining algorithm was designed to systematically analyze this wealth of information on SNPs, designed mutations, structural interactions, or functional roles of individual residues. Structurally corresponding positions in different CYPs were compared and universal selectivity-determining positions were identified. Based on the Cytochrome P450 Engineering Database (www.CYPED.BioCatNet.de) and a standard numbering scheme for all CYPs, 4000 residues in 168 CYPs mentioned in 2400 articles could be assigned to 440 structurally corresponding standard positions of the CYP fold, covering 96% of all standard positions. Seventeen individual standard positions were mentioned in the context of more than 32 different CYPs. The majority of these most frequently mentioned positions are located on the six substrate recognition sites and are involved in control of selectivity, such as the well-studied position 87 in CYP102A1 (P450(BM-3)) which was mentioned in the articles on 63 different CYPs. The recurrent citation of the 17 frequently mentioned positions for different CYPs suggests their universal functional relevance.

Conservation analysis of class-specific positions in cytochrome P450 monooxygenases: functional and structural relevance.

Cytochrome P450 monooxygenases (CYPs) constitute a ubiquitous, highly divergent protein family. Nevertheless, all CYPs share a common fold and conserved catalytic machinery. Based on the electron donor system, 10 classes of CYPs have been described, but most CYPs are members of class I accepting electrons from ferredoxin which is being reduced by FAD-containing reductase, or class II accepting electrons from FAD- and FMN-containing CPR-type reductase. Because of the low sequence conservation inside the two classes, the conserved class-specific positions are expected to be involved in aspects of electron transfer that are specific to the two types of reductases. In this work we present results from a conservation analysis of 16,732 CYP sequences derived from an updated version of the Cytochrome P450 Engineering Database (CYPED), using two class-specific numbering schemes. While no position was conserved on the distal, substrate-binding surface of the CYPs, several class-specific residues were found on the proximal, reductase-interacting surface; two class I-specific residues that were negatively charged, and three class II-specific residues that were aromatic or charged. The class-specific conservation of glycine and proline residues in the cysteine pocket indicates that there are class-specific differences in the flexibility of this element. Four heme-interacting arginines were conserved differently in each class, and a class-specific substitution of a heme-interacting tyrosine by histidine was found, pointing to a link between heme stabilization and the reductase type.