Photocatalytic C-O Coupling Enzymes That Operate via Intramolecular Electron Transfer.

Efficient and environmentally friendly conversion of light energy for direct utilization in chemical production has been a long-standing goal in enzyme design. Herein, we synthesized artificial photocatalytic enzymes by introducing an Ir photocatalyst and a Ni(bpy) complex to an optimal protein scaffold in close proximity. Consequently, the enzyme generated C-O coupling products with up to 96% yields by harvesting visible light and performing intramolecular electron transfer between the two catalysts. We systematically modulated the catalytic activities of the artificial photocatalytic cross-coupling enzymes by tuning the electrochemical properties of the catalytic components, their positions, and distances within a protein. As a result, we discovered the best-performing mutant that showed broad substrate scopes under optimized conditions. This work explicitly demonstrated that we could integrate and control both the inorganic and biochemical components of photocatalytic biocatalysis to achieve high yield and selectivity in valuable chemical transformations.

Underlying Role of Hydrophobic Environments in Tuning Metal Elements for Efficient Enzyme Catalysis.

The catalytic functions of metalloenzymes are often strongly correlated with metal elements in the active sites. However, dioxygen-activating nonheme quercetin dioxygenases (QueD) are found with various first-row transition-metal ions when metal swapping inactivates their innate catalytic activity. To unveil the molecular basis of this seemingly promiscuous yet metal-specific enzyme, we transformed manganese-dependent QueD into a nickel-dependent enzyme by sequence- and structure-based directed evolution. Although the net effect of acquired mutations was primarily to rearrange hydrophobic residues in the active site pocket, biochemical, kinetic, X-ray crystallographic, spectroscopic, and computational studies suggest that these modifications in the secondary coordination spheres can adjust the electronic structure of the enzyme-substrate complex to counteract the effects induced by the metal substitution. These results explicitly demonstrate that such noncovalent interactions encrypt metal specificity in a finely modulated manner, revealing the underestimated chemical power of the hydrophobic sequence network in enzyme catalysis.

Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB.

Graspetides, also known as ω-ester-containing peptides (OEPs), are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) bearing side chain-to-side chain macrolactone or macrolactam linkages. Here, we present the molecular details of precursor peptide recognition by the macrocyclase enzyme PsnB in the biosynthesis of plesiocin, a group 2 graspetide. Biochemical analysis revealed that, in contrast to other RiPPs, the core region of the plesiocin precursor peptide noticeably enhanced the enzyme-precursor interaction via the conserved glutamate residues. We obtained four crystal structures of symmetric or asymmetric PsnB dimers, including those with a bound core peptide and a nucleotide, and suggest that the highly conserved Arg213 at the enzyme active site specifically recognizes a ring-forming acidic residue before phosphorylation. Collectively, this study provides insights into the mechanism underlying substrate recognition in graspetide biosynthesis and lays a foundation for engineering new variants.

Symmetry-Adapted Synthesis of Dicopper Oxidases with Divergent Dioxygen Reactivity.

Artificial metalloenzymes have fed our understanding of how inorganic reactivities emerge, evolve, and diversify in protein environments. Herein, we created dinuclear copper oxidases by genetically encoding a metal-ligating unnatural amino acid (bpy-Ala) per protomer in the vicinity of the innate C2 rotational axis of a homo-oligomeric protein. The inherent protein symmetry allows the precise multiplication and placement of two Cu(bpy) species. Depending on the location of bpy-Ala, the tailor-made metalloenzymes exhibited electronically uncoupled or coupled dicopper sites. Consequently, they displayed various reactivities with dioxygen associated with multiple protons and electrons, illustrating a diverse chemical repertoire of artificial copper-dependent enzymes.

Folding of Circularly Permuted and Split Outer Membrane Protein F via Electrostatic Interactions with Terminal Residues.

Membrane proteins are essential targets in drug design, biosensing, and catalysis. In this study, we explored the folding of engineered outer membrane protein F (OmpF), an abundant and functional ß-barrel protein expressed in Gram-negative bacteria. We carried out circular permutation, splitting and self-complementation, and point mutation. The folding efficiency and kinetic analyses demonstrated that the N- and C-terminal residues of OmpF played critical roles in folding via electrostatic interactions with lipid headgroups. Our results indicate that native porins without charged terminal residues may be tightly downregulated to retain the integrity of the outer membrane, and this modification may facilitate the insertion and folding of modified membrane proteins under in vitro and in vivo conditions for various applications.

Emergence of metal selectivity and promiscuity in metalloenzymes.

Metal coordination with proteinaceous ligands has greatly expanded the chemical toolbox of proteins and their biological roles. The structure and function of natural metalloproteins have been determined according to the physicochemical properties of metal ions bound to the active sites. Concurrently, amino acid sequences are optimized for metal coordination geometry and/or dedicated action of metal ions in proteinaceous environments. In some occasions, however, natural enzymes exhibit promiscuous reactivity with more than one designated metal ion, under in vitro and/or in vivo conditions. In this review, we discuss selected examples of metalloenzymes that bind various first-row, mid- to late-transition metal ions for their native catalytic activities. From these examples, we suggest that environmental, inorganic, and biochemical factors, such as bioavailability, native organism, cellular compartment, reaction mechanism, binding affinity, protein sequence, and structure, might be responsible for determining metal selectivity or promiscuity. The current work proposes how natural metalloproteins might have emerged and adapted for specific metal incorporation under the given circumstances and may provide insights into the design and engineering of de novo metalloproteins.

Importance of Scaffold Flexibility/Rigidity in the Design and Directed Evolution of Artificial Metallo-ß-lactamases.

We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn4:C96RIDC14, which was constructed from cytochrome cb562 building blocks via a metal-templating strategy. Previously, we reported that Zn4:C96RIDC14 could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn8:A104AB34, which in turn displayed catalytic activity for the hydrolysis of activated esters and ß-lactam antibiotics. Zn8:A104AB34 was subsequently subjected to directed evolution via an in vivo selection strategy, leading to a variant Zn8:A104/G57AB34 which displayed enzyme-like Michaelis-Menten behavior for ampicillin hydrolysis. A criterion for the evolutionary utility or designability of a new protein structure is its ability to accommodate different active sites. With this in mind, we examined whether Zn4:C96RIDC14 could be tailored with alternative Zn coordination sites that could similarly display evolvable catalytic activities. We report here a detailed structural and functional characterization of new variant Zn8:AB54, which houses similar, unsaturated Zn coordination sites to those in Zn8:A104/G57AB34, but in completely different microenvironments. Zn8:AB54 displays Michaelis-Menten behavior for ampicillin hydrolysis without any optimization. Yet, the subsequent directed evolution of Zn8:AB54 revealed limited catalytic improvement, which we ascribed to the local protein rigidity surrounding the Zn centers and the lack of evolvable loop structures nearby. The relaxation of local rigidity via the elimination of adjacent disulfide linkages led to a considerable structural transformation with a concomitant improvement in ß-lactamase activity. Our findings reaffirm previous observations that the delicate balance between protein flexibility and stability is crucial for enzyme design and evolution.

Tracking a defined route for O2 migration in a dioxygen-activating diiron enzyme.

For numerous enzymes reactive toward small gaseous compounds, growing evidence indicates that these substrates diffuse into active site pockets through defined pathways in the protein matrix. Toluene/o-xylene monooxygenase hydroxylase is a dioxygen-activating enzyme. Structural analysis suggests two possible pathways for dioxygen access through the α-subunit to the diiron center: a channel or a series of hydrophobic cavities. To distinguish which is utilized as the O(2) migration pathway, the dimensions of the cavities and the channel were independently varied by site-directed mutagenesis and confirmed by X-ray crystallography. The rate constants for dioxygen access to the diiron center were derived from the formation rates of a peroxodiiron(III) intermediate, generated upon treatment of the diiron(II) enzyme with O(2). This reaction depends on the concentration of dioxygen to the first order. Altering the dimensions of the cavities, but not the channel, changed the rate of dioxygen reactivity with the enzyme. These results strongly suggest that voids comprising the cavities in toluene/o-xylene monooxygenase hydroxylase are not artifacts of protein packing/folding, but rather programmed routes for dioxygen migration through the protein matrix. Because the cavities are not fully connected into the diiron active center in the enzyme resting state, conformational changes will be required to facilitate dioxygen access to the diiron center. We propose that such temporary opening and closing of the cavities may occur in all bacterial multicomponent monooxygenases to control O(2) consumption for efficient catalysis. Our findings suggest that other gas-utilizing enzymes may employ similar structural features to effect substrate passage through a protein matrix.

Programming interchangeable and reversible heterooligomeric protein self-assembly using a bifunctional ligand.

Protein design for self-assembly allows us to explore the emergence of protein-protein interfaces through various chemical interactions. Heterooligomers, unlike homooligomers, inherently offer a comprehensive range of structural and functional variations. Besides, the macromolecular repertoire and their applications would significantly expand if protein components could be easily interchangeable. This study demonstrates that a rationally designed bifunctional linker containing an enzyme inhibitor and maleimide can guide the formation of diverse protein heterooligomers in an easily applicable and exchangeable manner without extensive sequence optimizations. As proof of concept, we selected four structurally and functionally unrelated proteins, carbonic anhydrase, aldolase, acetyltransferase, and encapsulin, as building block proteins. The combinations of two proteins with the bifunctional linker yielded four two-component heterooligomers with discrete sizes, shapes, and enzyme activities. Besides, the overall size and formation kinetics of the heterooligomers alter upon adding metal chelators, acidic buffer components, and reducing agents, showing the reversibility and tunability in the protein self-assembly. Given that the functional groups of both the linker and protein components are readily interchangeable, our work broadens the scope of protein-assembled architectures and their potential applications as functional biomaterials.

Discovery and Characterization of Polymyxin-Resistance Genes pmrE and pmrF from Sediment and Seawater Microbiome.

Polymyxins are the last-line antibiotics used to treat Gram-negative pathogens. Thus, the discovery and biochemical characterization of the resistance genes against polymyxins are urgently needed for diagnosis, treatment, and novel antibiotic design. Herein, we report novel polymyxin-resistance genes identified from sediment and seawater microbiome. Despite their low sequence identity against the known pmrE and pmrF, they show in vitro activities in UDP-glucose oxidation and l-Ara4N transfer to undecaprenyl phosphate, respectively, which occur as the part of lipid A modification that leads to polymyxin resistance. The expression of pmrE and pmrF also showed substantially high MICs in the presence of vanadate ions, indicating that they constitute polymyxin resistomes. IMPORTANCE Polymyxins are one of the last-resort antibiotics. Polymyxin resistance is a severe threat to combat multidrug-resistant pathogens. Thus, up-to-date identification and understanding of the related genes are crucial. Herein, we performed structure-guided sequence and activity analysis of five putative polymyxin-resistant metagenomes. Despite relatively low sequence identity to the previously reported polymyxin-resistance genes, at least four out of five discovered genes show reactivity essential for lipid A modification and polymyxin resistance, constituting antibiotic resistomes.

Design and directed evolution of noncanonical ß-stereoselective metalloglycosidases.

Metallohydrolases are ubiquitous in nearly all subclasses of hydrolases, utilizing metal elements to activate a water molecule and facilitate its subsequent dissociation of diverse chemical bonds. However, such a catalytic role of metal ions is rarely found with glycosidases that hydrolyze the glycosidic bonds in sugars. Herein, we design metalloglycosidases by constructing a hydrolytically active Zn-binding site within a barrel-shaped outer membrane protein OmpF. Structure- and mechanism-based redesign and directed evolution have led to the emergence of Zn-dependent glycosidases with catalytic proficiency of 2.8 × 109 and high ß-stereoselectivity. Biochemical characterizations suggest that the Zn-binding site constitutes a key catalytic motif along with at least one adjacent acidic residue. This work demonstrates that unprecedented metalloenzymes can be tailor-made, expanding the scope of inorganic reactivities in proteinaceous environments, resetting the structural and functional diversity of metalloenzymes, and providing the potential molecular basis of unidentified metallohydrolases and novel whole-cell biocatalysts.

Mechanistic studies of reactions of peroxodiiron(III) intermediates in T201 variants of toluene/o-xylene monooxygenase hydroxylase.

Site-directed mutagenesis studies of a strictly conserved T201 residue in the active site of toluene/o-xylene monooxygenase hydroxylase (ToMOH) revealed that a single mutation can facilitate kinetic isolation of two distinctive peroxodiiron(III) species, designated T201(peroxo) and ToMOH(peroxo), during dioxygen activation. Previously, we characterized both oxygenated intermediates by UV-vis and Mössbauer spectroscopy, proposed structures from DFT and QM/MM computational studies, and elucidated chemical steps involved in dioxygen activation through the kinetic studies of T201(peroxo) formation. In this study, we investigate the kinetics of T201(peroxo) decay to explore the reaction mechanism of the oxygenated intermediates following O(2) activation. The decay rates of T201(peroxo) were monitored in the absence and presence of external (phenol) or internal (tryptophan residue in an I100W variant) substrates under pre-steady-state conditions. Three possible reaction models for the formation and decay of T201(peroxo) were evaluated, and the results demonstrate that this species is on the pathway of arene oxidation and appears to be in equilibrium with ToMOH(peroxo).

Multiple roles of component proteins in bacterial multicomponent monooxygenases: phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas sp. OX1.

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H(2)O(2)) generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was ∼50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H(2)O(2) formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.

Insights into the different dioxygen activation pathways of methane and toluene monooxygenase hydroxylases.

The methane and toluene monooxygenase hydroxylases (MMOH and TMOH, respectively) have almost identical active sites, yet the physical and chemical properties of their oxygenated intermediates, designated P*, H(peroxo), Q, and Q* in MMOH and ToMOH(peroxo) in a subclass of TMOH, ToMOH, are substantially different. We review and compare the structural differences in the vicinity of the active sites of these enzymes and discuss which changes could give rise to the different behavior of H(peroxo) and Q. In particular, analysis of multiple crystal structures reveals that T213 in MMOH and the analogous T201 in TMOH, located in the immediate vicinity of the active site, have different rotatory configurations. We study the rotational energy profiles of these threonine residues with the use of molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) computational methods and put forward a hypothesis according to which T213 and T201 play an important role in the formation of different types of peroxodiiron(III) species in MMOH and ToMOH. The hypothesis is indirectly supported by the QM/MM calculations of the peroxodiiron(III) models of ToMOH and the theoretically computed Mössbauer spectra. It also helps explain the formation of two distinct peroxodiiron(III) species in the T201S mutant of ToMOH. Additionally, a role for the ToMOD regulatory protein, which is essential for intermediate formation and protein functioning in the ToMO system, is advanced. We find that the low quadrupole splitting parameter in the Mössbauer spectrum observed for a ToMOH(peroxo) intermediate can be explained by protonation of the peroxo moiety, possibly stabilized by the T201 residue. Finally, similarities between the oxygen activation mechanisms of the monooxygenases and cytochrome P450 are discussed.

Design of artificial metalloenzymes with multiple inorganic elements: The more the merrier.

A large fraction of metalloenzymes harbors multiple metal-centers that are electronically and/or functionally arranged within their proteinaceous environments. To explore the orchestration of inorganic and biochemical components and to develop bioinorganic catalysts and materials, we have described selected examples of artificial metalloproteins having multiple metallocofactors that were grouped depending on their initial protein scaffolds, the nature of introduced inorganic moieties, and the method used to propagate the number of metal ions within a protein. They demonstrated that diverse inorganic moieties can be selectively grafted and modulated in protein environments, providing a retrosynthetic bottom-up approach in the design of versatile and proficient biocatalysts and biomimetic model systems to explore fundamental questions in bioinorganic chemistry.

Symmetry-related residues as promising hotspots for the evolution of de novo oligomeric enzymes.

Directed evolution has provided us with great opportunities and prospects in the synthesis of tailor-made proteins. It, however, often requires at least mid to high throughput screening, necessitating more effective strategies for laboratory evolution. We herein demonstrate that protein symmetry can be a versatile criterion for searching for promising hotspots for the directed evolution of de novo oligomeric enzymes. The randomization of symmetry-related residues located at the rotational axes of artificial metallo-ß-lactamase yields drastic effects on catalytic activities, whereas that of non-symmetry-related, yet, proximal residues to the active site results in negligible perturbations. Structural and biochemical analysis of the positive hits indicates that seemingly trivial mutations at symmetry-related spots yield significant alterations in overall structures, metal-coordination geometry, and chemical environments of active sites. Our work implicates that numerous artificially designed and natural oligomeric proteins might have evolutionary advantages of propagating beneficial mutations using their global symmetry.

Genomic Determinants Encode the Reactivity and Regioselectivity of Flavin-Dependent Halogenases in Bacterial Genomes and Metagenomes.

Halogenases create diverse natural products by utilizing halide ions and are of great interest in the synthesis of potential pharmaceuticals and agrochemicals. An increasing number of halogenases discovered in microorganisms are annotated as flavin-dependent halogenases (FDHs), but their chemical reactivities are markedly different and the genomic contents associated with such functional distinction have not been revealed yet. Even though the reactivity and regioselectivity of FDHs are essential in the halogenation activity, these FDHs are annotated inaccurately in the protein sequence repositories without characterizing their functional activities. We carried out a comprehensive sequence analysis and biochemical characterization of FDHs. Using a probabilistic model that we built in this study, FDHs were discovered from 2,787 bacterial genomes and 17 sediment metagenomes. We analyzed the essential genomic determinants that are responsible for substrate binding and subsequent reactions: four flavin adenine dinucleotide-binding, one halide-binding, and four tryptophan-binding sites. Compared with previous studies, our study utilizes large-scale genomic information to propose a comprehensive set of sequence motifs that are related to the active sites and regioselectivity. We reveal that the genomic patterns and phylogenetic locations of the FDHs determine the enzymatic reactivities, which was experimentally validated in terms of the substrate scope and regioselectivity. A large portion of publicly available FDHs needs to be reevaluated to designate their correct functions. Our genomic models establish comprehensive links among genotypic information, reactivity, and regioselectivity of FDHs, thereby laying an important foundation for future discovery and classification of novel FDHs. IMPORTANCE Halogenases are playing an important role as tailoring enzymes in biosynthetic pathways. Flavin-dependent tryptophan halogenases (Trp-FDHs) are among the enzymes that have broad substrate scope and high selectivity. From bacterial genomes and metagenomes, we found highly diverse halogenase sequences by using a well-trained profile hidden Markov model built from the experimentally validated halogenases. The characterization of genotype, steady-state activity, substrate scope, and regioselectivity has established comprehensive links between the information encoded in the genomic sequence and reactivity of FDHs reported here. By constructing models for accurate and detailed sequence markers, our work should guide future discovery and classification of novel FDHs.

Active site threonine facilitates proton transfer during dioxygen activation at the diiron center of toluene/o-xylene monooxygenase hydroxylase.

Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was explored by stopped-flow UV-vis and Mössbauer spectroscopy. These studies demonstrate that the dioxygen activation mechanism is preserved in all T201 variants; however, both the formation and decay kinetics of a peroxodiiron(III) intermediate, T201(peroxo), were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O(2) activation in the T201S, T201C, and T201G variants under various reaction conditions revealed that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. A mechanism is postulated for dioxygen activation, and possible structures of oxygenated intermediates are discussed.