On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non-Native Halogenases.

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (FlC4a-OOH), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H2O2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that FlC4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from FlC4a-OOH cannot halogenate their substrates. Remarkably, in situ H2O2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming FlC4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation.

Unifying and versatile features of flavin-dependent monooxygenases: Diverse catalysis by a common C4a-(hydro)peroxyflavin.

Flavin-dependent monooxygenases (FDMOs) are known for their remarkable versatility and for their crucial roles in various biological processes and applications. Extensive research has been conducted to explore the structural and functional relationships of FDMOs. The majority of reported FDMOs utilize C4a-(hydro)peroxyflavin as a reactive intermediate to incorporate an oxygen atom into a wide range of compounds. This review discusses and analyzes recent advancements in our understanding of the structural and mechanistic features governing the enzyme functions. State-of-the-art discoveries related to common and distinct structural properties governing the catalytic versatility of the C4a-(hydro)peroxyflavin intermediate in selected FDMOs are discussed. Specifically, mechanisms of hydroxylation, dehalogenation, halogenation, and light-emitting reactions by FDMOs are highlighted. We also provide new analysis based on the structural and mechanistic features of these enzymes to gain insights into how the same intermediate can be harnessed to perform a wide variety of reactions. Challenging questions to obtain further breakthroughs in the understanding of FDMOs are also proposed.

Enzymes, In Vivo Biocatalysis, and Metabolic Engineering for Enabling a Circular Economy and Sustainability.

Since the industrial revolution, the rapid growth and development of global industries have depended largely upon the utilization of coal-derived chemicals, and more recently, the utilization of petroleum-based chemicals. These developments have followed a linear economy model (produce, consume, and dispose). As the world is facing a serious threat from the climate change crisis, a more sustainable solution for manufacturing, i.e., circular economy in which waste from the same or different industries can be used as feedstocks or resources for production offers an attractive industrial/business model. In nature, biological systems, i.e., microorganisms routinely use their enzymes and metabolic pathways to convert organic and inorganic wastes to synthesize biochemicals and energy required for their growth. Therefore, an understanding of how selected enzymes convert biobased feedstocks into special (bio)chemicals serves as an important basis from which to build on for applications in biocatalysis, metabolic engineering, and synthetic biology to enable biobased processes that are greener and cleaner for the environment. This review article highlights the current state of knowledge regarding the enzymatic reactions used in converting biobased wastes (lignocellulosic biomass, sugar, phenolic acid, triglyceride, fatty acid, and glycerol) and greenhouse gases (CO2 and CH4) into value-added products and discusses the current progress made in their metabolic engineering. The commercial aspects and life cycle assessment of products from enzymatic and metabolic engineering are also discussed. Continued development in the field of metabolic engineering would offer diversified solutions which are sustainable and renewable for manufacturing valuable chemicals.

Dissecting the low catalytic capability of flavin-dependent halogenases.

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I-, Br-, and Cl- but not F- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.

Structures, mechanisms and applications of flavin-dependent halogenases.

Overall, this review highlights the structures, mechanisms and applications of flavin-dependent halogenases (FDHs) for future development of FDHs as potential biocatalysts. FDHs catalyze incorporation of halogen atoms into a broad range of substrates. The reactions involved in the production of various halogenated natural products which are important drugs. Typical substrates for FDHs include indole, pyrrole, phenolic and aliphatic compounds. In addition to organic substrates, all FDHs utilize reduced FAD (FADH-), oxygen and halides as co-substrates. Structural studies reveal that FDHs all have similar FAD binding sites. However, FDHs have variations between the different isotypes including different recognition residues for substrate binding and some unique loop structures and conformations. These different structural differences suggest that variations in reaction catalysis exist. However, limited knowledge of the reaction mechanisms of FDHs is currently available. Various biocatalytic applications of FDHs have been explored. Further investigation of the catalytic reactions of FDHs is essential for improving enzyme engineering work to enable FDHs catalysis of challenging reactions.