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DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

Reductions play a key role in organic synthesis, producing chiral products with new functionalities. Enzymes can catalyse such reactions with exquisite stereo‐, regio‐ and chemoselectivity, leading the way to alternative shorter classical synthetic routes towards not only high‐added‐value compounds but also bulk chemicals. In this review we describe the synthetic state‐of‐the‐art and potential of enzymes that catalyse reductions, ranging from carbonyl, enone and aromatic reductions to reductive aminations. [ABSTRACT FROM AUTHOR]

Reduktionsreaktionen spielen eine zentrale Rolle in der organischen Chemie zur Synthese chiraler Produkte. Insbesondere Enzyme katalysieren solche Reaktionen in hoher Stereo‐, Regio‐ und Chemoselektivität. Somit eröffnen sie kürzere Alternativrouten zur Synthese von Spezial‐ und Bulkchemikalien. Dieser Aufsatz beschreibt den aktuellen Stand, die Herausforderungen und Möglichkeiten enzymkatalysierter Reduktionen von (konjugierten) Carbonylverbindungen, Aromaten sowie der reduktiven Aminierung. [ABSTRACT FROM AUTHOR]

Old yellow enzymes are able to catalyze asymmetric C=C reductions. A mediated electroenzymatic process to regenerate the NADPH in combination with an old yellow enzyme was investigated. Due to the fact that the overall process was affected by a broad set of parameters, a design of experiments (DoE) approach was chosen to identify suitable process conditions. Process conditions with high productivities of up to 2.27 mM/h in combination with approximately 90% electron transfer efficiency were identified. [ABSTRACT FROM AUTHOR]

The one-pot conversion of cycloalkanes to their corresponding lactones was achieved through the use of a synthetic pathway consisting of a cytochrome P450 monooxygenase (CYP450) for initial oxyfunctionalization of the cycloalkane, an alcohol dehydrogenase for ketone production and a Baeyer-Villiger monooxygenase for lactone formation. Through variation of the cofactor dependence of the biocatalysts and the cofactor regeneration system, final product concentrations of nearly 3 g L−1 enantholactone (2-oxocanone) from cycloheptane was reached within 12 h with a total turnover number (TTN) of 4185 with respect to the CYP450. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs) of the CYP153 family catalyse terminal hydroxylation of n-alkanes. Alkane hydroxylating mutants of self-sufficient CYP102A1 have also been described. We evaluated two CYP153s (a three-component system and a fused self-sufficient CYP), wild-type CYP102A1 and nine CYP102A1 mutants, for the conversion of three cycloalkanes (C6, C7 and C8) and three n-alkanes (C6, C8 and C10) using whole cells (WCs) and crude cell-free extracts (CFEs). The aim was to identify substrate-enzyme combinations that give high product titres and space-time yields (STYs). Comparisons were made using total turnover numbers (TTNs) and turnover frequencies (TOFs) to normalize for CYP expression. Reactions were carried out using high enzyme and substrate concentrations compatible with high STYs. Under these conditions CYP102A1 and the double R47L,Y51F mutant, although not regioselective, performed better on all substrates in terms of product titres over 8 h, and thus STYs and TTNs, than heavily mutated variants that have been reported to give very high TOFs. CYP153A6, with its ferredoxin (Fdx) and ferredoxin reductase (FdR), emerged as the superior catalyst for conversion of n-alkanes. In addition to its excellent regioselectivity it also gave the highest final product titres and STYs in WC conversions of hexane and octane. Interaction with FdR and Fdx initially limited performance in CFEs, but with additional FdR and Fdx gave 1-octanol titres of 50 mmol⋅LBRM−1 and TTNs exceeding 12,000 over 18 h, rivalling results reported with self-sufficient CYPs. Selecting biocatalysts for application requires caution, since experimental conditions such as amount of substrate added and solubility as well as cofactor dependence and regeneration can have a profound effect on catalyst performance, while stability and efficiency with regard to cofactor usage (coupling efficiency) are at least as important as TOFs when high product titres and STYs are the target. [ABSTRACT FROM AUTHOR]

The article discusses the chemoenzymatic reduction processes for conjugated C=C double bonds. The authors used organometallic complex pentamethyl cyclopentadienyl rhodium bipyridine as an alternative to enoate reductases which are commonly applied in chemical synthesis. According to them, the ability of the complex to regenerate reduced flavin cofactors is helpful for the regeneration of the reductases.

Continuous low-level supply or in situ generation of hydrogen peroxide (H2O2) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C–H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO2 or carbon have sufficient activity at ambient temperature and pressure to generate H2O2 from H2 and O2 and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled. Continuous low-level supply or in situ generation of hydrogen peroxide (H2O2) is essential for the stability of unspecific peroxygenases. Here, the authors demonstrate that AuPd / TiO2 can generate sufficient H2O2 for the engineered unspecific heme-thiolate peroxygenase PaDa-I to oxidise a range of C-H bonds. [ABSTRACT FROM AUTHOR]

Redox reactions catalyzed by highly selective nicotinamide-dependent oxidoreductases are rising to prominence in industry. The cost of nicotinamide adenine dinucleotide coenzymes has led to the use of well-established elaborate regeneration systems and more recently alternative synthetic biomimetic cofactors. These biomimetics are highly attractive to use with ketoreductases for asymmetric catalysis. In this work, we show that the commonly studied cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) can be used with alcohol dehydrogenases (ADHs) under certain conditions. First, we carried out the rhodium-catalyzed recycling of BNAH with horse liver ADH (HLADH), observing enantioenriched product only with unpurified enzyme. Then, a series of cell-free extracts and purified ketoreductases were screened with BNAH. The use of unpurified enzyme led to product formation, whereas upon dialysis or further purification no product was observed. Several other biomimetics were screened with various ADHs and showed no or very low activity, but also no inhibition. BNAH as a hydride source was shown to directly reduce nicotinamide adenine dinucleotide (NAD) to NADH. A formate dehydrogenase could also mediate the reduction of NAD from BNAH. BNAH was established to show no or very low activity with ADHs and could be used as a hydride donor to recycle NADH. [ABSTRACT FROM AUTHOR]

Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer–Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability. [ABSTRACT FROM AUTHOR]