Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase

Biocatalytic dealkylation of aryl methyl ethers is an attractive reaction for valorization of lignin components, as well as for deprotection of hydroxy functionalities in synthetic chemistry. We explored the demethylation of various aryl methyl ethers by using an oxidative demethylase from Pseudomonas sp. HR199. The Rieske monooxygenase VanA and its partner electron transfer protein VanB were recombinantly coexpressed in Escherichia coli and they constituted at least 25 % of the total protein content. Enzymatic transformations showed that VanB accepts NADH and NADPH as electron donors. The VanA–VanB system demethylates a number of aromatic substrates, the presence of a carboxylic acid moiety is essential, and the catalysis occurs selectively at the meta position to this carboxylic acid in the aromatic ring. The reaction is inhibited by the by-product formaldehyde. Therefore, we tested three different cascade/tandem reactions for cofactor regeneration and formaldehyde elimination; in particular, conversion was improved by addition of formaldehyde dehydrogenase and formate dehydrogenase. Finally, the biocatalyst was applied for the preparation of protocatechuic acid from vanillic acid, giving a 77 % yield of the desired product. The described reaction may find application in the conversion of lignin components into diverse hydroxyaromatic building blocks and generally offers potential for new, mild methods for efficient unmasking of phenols.     

In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.     

Protein Engineering of the Progesterone Hydroxylating P450‐Monooxygenase CYP17A1 Alters Its Regioselectivity

The CYP171 enzyme is known to catalyse a key step in the steroidogenesis of mammals. The substrates progesterone and pregnenolone are first hydroxylated at the C17 position, and this is followed by cleavage of the C17?C20 bond to yield important precursors for glucosteroids and androgens. In this study, we focused on the reaction of the bovine CYP17A1 enzyme with progesterone as a substrate. On the basis of a created homology model, active‐site residues were identified and systematically mutated to alanine. In whole‐cell biotransformations, the importance of the N202, R239, G297 and E305 residues for substrate conversion was confirmed. Additionally, mutation of the L206, V366 and V483 residues enhanced the formation of the 16α‐hydroxyprogesterone side product up to 40 % of the total product formation. Furthermore, residue L105 was found not to be involved in this side activity, which contradicts a previous study with the human enzyme.     

The Oxidation of Hydrophobic Aromatic Substrates by Using a Variant of the P450 Monooxygenase CYP101B1

The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency than norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which, in the latter enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was mutated to phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. The H85F mutant lowered the binding affinity and activity of the enzyme for β-ionone and altered the oxidation selectivity. This variant also showed enhanced affinity and activity towards alkylbenzenes, styrenes and methylnaphthalenes. For example the rate of product formation for acenaphthene oxidation was improved sixfold to 245 nmol per nmol CYP per min. Certain disubstituted naphthalenes and substrates, such as phenylcyclohexane and biphenyls, were oxidised with lower activity by the H85F variant. Variants at H85 (A and G) designed to introduce additional space into the active site so as to accommodate these larger substrates did not improve the oxidation activity. As the H85F mutant of CYP101B1 improved the oxidation of hydrophobic substrates, this residue is likely to be in the substrate binding pocket or the access channel of the enzyme. The side chain of the histidine might interact with the carbonyl groups of the favoured norisoprenoid substrates of CYP101B1.     

Artificial reconstruction of two cryptic angucycline antibiotic biosynthetic pathways

Genome-sequencing projects have revealed that Streptomyces bacteria have the genetic potential to produce considerably larger numbers of natural products than can be observed under standard laboratory conditions. Cryptic angucycline-type aromatic polyketide gene clusters are particularly abundant. Sequencing of two such clusters from Streptomyces sp. PGA64 and H021 revealed the presence of several open reading frames that could be involved in processing the basic angucyclic carbon skeleton. The pga gene cluster contains one putative FAD-dependant monooxygenase (pgaE) and a putatively bifunctional monooxygenase/short chain alcohol reductase (pgaM), whereas the cab cluster contains two similar monooxygenases (cabE and cabM) and an independent reductase (cabV). In this study we have reconstructed the biosynthetic pathways for aglycone synthesis by cloning and sequentially expressing the angucycline tailoring genes with genes required for the synthesis of the unmodified angucycline metabolite-UWM6-in Streptomyces lividans TK24. The expression studies unequivocally showed that, after the production of UWM6, the pathways proceed through the action of the similar monooxygenases PgaE and CabE, followed by reactions catalysed by PgaM and CabMV. Analysis of the metabolites produced revealed that addition of pgaE and cabE genes directs both pathways to a known shunt product, rabelomycin, whereas expression of all genes from a given pathway results in the production of the novel angucycline metabolites gaudimycin A and B. However, one of the end products is most probably further modified by endogenous S. lividans TK24 enzymes. These experiments demonstrate that genes that are either inactive or cryptic in their native host can be used as biosynthetic tools to generate new compounds.     

Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Baeyer–Villiger monooxygenase (BVMO)‐mediated regiodivergent conversions of asymmetric ketones can lead to the formation of “normal” or “abnormal” lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active‐site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2‐oxo‐Δ³‐4,5,5‐trimethylcyclopentenylacetyl‐CoA monooxygenase from Pseudomonas putida (OTEMO) for cis‐bicyclo[3.2.0]hept‐2‐en‐6‐one ( 1 ) and trans‐dihydrocarvone ( 2 ), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild‐type enzyme converted (?)‐ 1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)‐ 2 , whereas the wild‐type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO_Arthro and CHMO_Acineto) to switch their regioselectivity towards (+)‐ 2 , which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.     

Effect of Two Wheat Cultivars Differing in Hydroxamic Acid Concentration on Detoxification Metabolism in the AphidSitobion avenae

Hydroxamic acids (Hx) are wheat secondary metabolites conferring resistance for cereals against aphids. The activity of five enzymatic systems were evaluated in the aphid Sitobion avenae reared on the high-Hx wheat cultivar Chagual and the low-Hx wheat cultivar Huayún for 10 generations. Enzyme solutions were prepared from aphid homogenates and assayed for mixed function oxidases (including cytochrome P-450 monooxygenases and NADPH cytochrome c reductase), glutathione S-transferases, esterases, and catalase. Specific activities per aphid individual of cytochrome P-450 monooxygenases, NADPH cytochrome c reductase, glutathione S-transferases, and esterases were significantly increased in wheat cultivars relative to oat (only marginal increase of esterases in Chagual). Aphids fed on cv. Huayún showed an overall higher induction of enzymatic systems than those fed on cv. Chagual. Comparison of these results with reported effects of Hx on detoxifying enzymes in other insects, including aphids, support the hypothesis that these enzymatic pathways play an important role in the detoxification of toxic host-plant secondary metabolites.     

Protein engineering of toluene monooxygenases for synthesis of hydroxytyrosol

Hyroxytyrosol (HTyr), an important phenol present in olives, stands out as a compound of high added value due to its exceptional antioxidant, antimicrobial and anticarcinogenic activities. This work describes the synthesis of HTyr via double hydroxylation of 2-phenylethanol (PEA) employing toluene monooxygenases (TMOs) as biocatalysts. Wild-type TMOs were initially evaluated for their ability to oxidise PEA and structurally-related substrates, providing better understanding of the factors responsible for controlling the regiospecificity. Both the length of the alkyl side chain and the presence of the hydroxyl group were found to influence the activity, possibly by interfering with the substrates’ entrance into the active site. Directed evolution of toluene-4-monooxygenase of Pseudomonas mendocina KR1 led to the discovery of variant TmoA S395C with a 15-fold increase in PEA hydroxylation rate. Saturation-mutagenesis at position TmoA I100 resulted in the finding of novel HTyr-producing variants I100A, I100S and I100G.     

Unveiling Two Consecutive Hydroxylations: Mechanisms of Aromatic Hydroxylations Catalyzed by Flavin-Dependent Monooxygenases for the Biosynthesis of Actinorhodin and Related Antibiotics

Flavin-dependent monooxygenases are ubiquitous in living systems and are classified into single- or two-component systems. Actinorhodin, produced by Streptomyces coelicolor, is a representative polycyclic polyketide that is hydroxylated through the action of the two-component ActVA-5/ActVB hydroxylase system. These homologous systems are widely distributed in bacteria, but their reaction mechanisms remain unclear. This in vitro investigation has provided chemical proof of two consecutive hydroxylations via hydroxynaphthalene intermediates involved in actinorhodin biosynthesis. The ActVA-5 oxygenase component catalyzed a stepwise dihydroxylation of the substrate, whereas the ActVB flavin reductase not only supplied a reduced cofactor, but also regulated the quinone–hydroquinone interconversion of an intermediate. Our study provides clues for understanding the general biosynthetic mechanisms of highly functionalized aromatic natural products with structural diversity.