Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes.

Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O2 mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states. The physical and chemical principles of flavin-based chemistry have been investigated widely. In the following pages we summarize the state of the art on a key area of research in flavin enzymology: the molecular basis for the activation of O2 by flavin-dependent oxidases and monooxygenases. In general terms, oxidases use O2 as an electron acceptor to produce H2O2, while monooxygenases activate O2 by forming a flavin intermediate and insert an oxygen atom into the substrate. First, we analyze how O2 reaches the flavin cofactor embedded in the protein matrix through dedicated access pathways. Then we approach O2 activation from the perspective of the monooxygenases, their preferred intermediate, the C(4a)-(hydro)peroxyflavin, and the cases in which other intermediates have been described. Finally, we focus on understanding how the architectures developed in the active sites of oxidases promote O2 activation and which other factors operate in its reactivity.

The crystal structure of P450-TT heme-domain provides the first structural insights into the versatile class VII P450s.

The first crystal structure of a class VII P450, CYP116B46 from Tepidiphilus thermophilus, has been solved at 1.9 Šresolution. The structure reveals overall conservation of the P450-fold and a water conduit around the I-helix. Active site residues have been identified and sequence comparisons have been made with other class VII enzymes. A structure similarity search demonstrated that the P450-TT structure is similar to enzymes capable of oxy-functionalization of fatty acids, terpenes, macrolides, steroids and statins. The insight gained from solving this structure will provide a guideline for future engineering and modelling studies on this catalytically promiscuous class of enzymes.

Polycyclic Ketone Monooxygenase from the Thermophilic Fungus Thermothelomyces thermophila: A Structurally Distinct Biocatalyst for Bulky Substrates.

Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.

Use of Methylmalonyl-CoA Epimerase in Enhancing Crotonase Stereoselectivity.

The use of methylmalonyl-CoA epimerase (MCEE) to improve stereoselectivity in crotonase-mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl-CoA carboxylase reductase and carboxymethylproline synthase in a three-enzyme one-pot sequential synthesis of functionalised C-5 carboxyalkylprolines starting from crotonyl-CoA and carbon dioxide.

The enzymes of ß-lactam biosynthesis.

The ß-lactam antibiotics and related ß-lactamase inhibitors are amongst the most important small molecules in clinical use. Most, but not all, ß-lactams including penicillins, cephalosporins, and clavulanic acid are produced via fermentation or via modification of fermented intermediates, with important exceptions being the carbapenems and aztreonam. The desire for more efficient routes to existing antibiotics and for access to new and synthetically challenging ones stimulates continued interest in ß-lactam biosynthesis. We review knowledge of the pathways leading to ß-lactam antibiotics focusing on the mechanisms, structures and biocatalytic applications of the enzymes involved.

Stereoselective preparation of lipidated carboxymethyl-proline/pipecolic acid derivatives via coupling of engineered crotonases with an alkylmalonyl-CoA synthetase.

The trisubstituted enolate- and C-C bond-forming capacities of engineered carboxymethylproline synthases CMPSs are coupled with the malonyl-CoA synthetase MatB to enable stereoselective preparation of 5- and 6-membered N-heterocycles functionalised with alkyl-substituted carboxymethyl side chains, starting from achiral alkyl-substituted malonic acids and L-amino acid semialdehydes. The results illustrate the biocatalytic utility of crotonases in tandem enzyme-catalysed reactions for stereoselective synthesis.

Crotonase catalysis enables flexible production of functionalized prolines and carbapenams.

The biocatalytic versatility of wildtype and engineered carboxymethylproline synthases (CMPSs) is demonstrated by the preparation of functionalized 5-carboxymethylproline derivatives methylated at C-2, C-3, C-4, or C-5 of the proline ring from appropriately substituted amino acid aldehydes and malonyl-coenzyme A. Notably, compounds with a quaternary center (at C-2 or C-5) were prepared in a stereoselective fashion by engineered CMPSs. The substituted-5-carboxymethyl-prolines were converted into the corresponding bicyclic ß-lactams using a carbapenam synthetase. The results demonstrate the utility of the crotonase superfamily enzymes for stereoselective biocatalysis, the amenability of carbapenem biosynthesis pathways to engineering for the production of new bicyclic ß-lactam derivatives, and the potential of engineered biocatalysts for the production of quaternary centers.

Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering.

Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.

Red Lapacho (Tabebuia impetiginosa)--a global ethnopharmacological commodity?

Red Lapacho (Tabebuia impetiginosa, syn. Tabebuia avellanedae), a canopy tree indigenous to the Amazonian rainforest and other parts of South America, has been acclaimed to be one of the "miraculous" cures for cancer and tumours. For the first time, during the 1960s, it attracted considerable attention in Brazil and Argentina as a 'wonder drug'. Traditionally, the botanical drug is widely used in local and traditional phytomedicine, usually ingested as a decoction prepared from the inner bark of the tree to treat numerous conditions like bacterial and fungal infections, fever, syphilis, malaria, trypanosomiasis, as well as stomach and bladder disorders. As early as 1873, biomedical uses of Red Lapacho ("Pau D'Arco") were reported. In 1967 after reports in the Brazilian press it came back to the light of clinicians (and the public in general). The news magazine O'Cruzeiro started reporting "miraculous" cures in cancer patients in a hospital. Natural sciences interest in the plant also began in the 1960s when the United States National Cancer Institute (NCI) systematically began researching plant extracts all over the world looking for active compounds against cancer and looked at Tabebuia impetiginosa in considerable detail. Two main bioactive components have been isolated from Tabebuia impetiginosa: lapachol and beta-lapachone. beta-Lapachone is considered to be the main anti-tumour compound, and pro-apoptotic effects were observed in vitro. Some mechanistic studies on this compound's molecular effects have been conducted. The other main constituents isolated from Red Lapacho are also reviewed briefly. The drug appears to be generally safe and one of the most important interactions of Tabebuia impetiginosa has been associated with interference in the biological cycle of Vitamin K in the body. The botanical (drug) material available on the international markets seems to be of varying quality and composition, making a specific assessment of the products' therapeutic claims problematic. This also highlights the need for appropriate analytical techniques, which are reviewed as well. The bioscientific evidence for products derived from Tabebuia impetiginosa is insufficient and one of the core challenges of future research will be--based on the recognition of the drug's widespread use--to establish appropriate quality control procedures. Further research into the clinical effects and the pharmacology of chemically characterized extracts is also warranted.

Biocatalytic production of bicyclic ß-lactams with three contiguous chiral centres using engineered crotonases.

There is a need to develop asymmetric routes to functionalised ß-lactams, which remain the most important group of antibacterials. Here we describe biocatalytic and protein engineering studies concerning carbapenem biosynthesis enzymes, aiming to enable stereoselective production of functionalised carbapenams with three contiguous chiral centres. Structurally-guided substitutions of wildtype carboxymethylproline synthases enable tuning of their C-N and C-C bond forming capacity to produce 5-carboxymethylproline derivatives substituted at C-4 and C-6, from amino acid aldehyde and malonyl-CoA derivatives. Use of tandem enzyme incubations comprising an engineered carboxymethylproline synthase and an alkylmalonyl-CoA forming enzyme (i.e. malonyl-CoA synthetase or crotonyl-CoA carboxylase reductase) can improve stereocontrol and expand the product range. Some of the prepared 4,6-disubstituted-5-carboxymethylproline derivatives are converted to bicyclic ß-lactams by carbapenam synthetase catalysis. The results illustrate the utility of tandem enzyme systems involving engineered crotonases for asymmetric bicyclic ß-lactam synthesis.

Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme.

Detoxifying enzymes such as flavin-containing monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question. Here, we addressed this issue by applying a cumulative alanine mutagenesis approach to cyclohexanone monooxygenase from Thermocrispum municipale, a flavin-dependent Baeyer-Villiger monooxygenase which we chose as a model system because of its pronounced thermostability and substrate promiscuity. Simultaneous removal of up to eight noncatalytic active-site side chains including four phenylalanines had no effect on protein folding, thermostability, and cofactor loading. We observed a linear decrease in activity, rather than a selectivity switch, and attributed this to a less efficient catalytic environment in the enlarged active-site space. Time-resolved kinetic studies confirmed this interpretation. We also determined the crystal structure of the enzyme in complex with a mimic of the reaction intermediate that shows an unaltered overall protein conformation. These findings led us to propose that this cyclohexanone monooxygenase may lack a distinct substrate selection mechanism altogether. We speculate that the main or exclusive function of the protein shell in promiscuous enzymes might be the stabilization and accessibility of their very reactive catalytic intermediates.

Stereoselective C-C bond formation catalysed by engineered carboxymethylproline synthases.

The reaction of enol(ate)s with electrophiles is used extensively in organic synthesis for stereoselective C-C bond formation. Protein-based catalysts have had comparatively limited application for the stereoselective formation of C-C bonds of choice via enolate chemistry. We describe protein engineering studies on 5-carboxymethylproline synthases, members of the crotonase superfamily, aimed at enabling stereoselective C-C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates. Active site substitutions, including at the oxyanion binding site, enable the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The results reveal the potential of the ubiquitous crotonase superfamily as adaptable catalysts for the control of enolate chemistry.