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.

Advancement in fabrication of carbon nanotube tip for atomic force microscope using multi-axis nanomanipulator in scanning electron microscope.

We have constructed a new nanomanipulator (NM) in a field emission scanning electron microscope (FE-SEM) to fabricate carbon nanotube (CNT) tip to precisely adjust the length and attachment angle of CNT onto the mother atomic force microscope (AFM) tip. The new NM is composed of 2 modules, each of which has the degree of freedom of three-dimensional rectilinear motionx,yandzand one-dimensional rotational motionθ. The NM is mounted on the stage of a FE-SEM. With the system of 14 axes in total which includes 5 axes of FE-SEM and 9 axes of nano-actuators, it was possible to see CNT tip from both rear and side view about the mother tip. With the help of new NM, the attachment angle error could be reduced down to 0° as seen from both the side and the rear view, as well as, the length of the CNT could be adjusted with the precision using electron beam induced etching. For the proper attachment of CNT on the mother tip surface, the side of the mother tip was milled with focused ion beam. In addition, electron beam induced deposition was used to strengthen the adhesion between CNT and the mother tip. In order to check the structural integrity of fabricated CNT, transmission electron microscope image was taken which showed the fine cutting of CNT and the clean surface as well. Finally, the performance of the fabricated CNT tip was demonstrated by imaging 1-D grating and DNA samples with atomic force microscope in tapping mode.

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.

Current biochemical understanding regarding the metabolism of acinetobactin, the major siderophore of the human pathogen Acinetobacter baumannii, and outlook for discovery of novel anti-infectious agents based thereon.

Covering: 1994 to 2019Owing to the rapid increase in nosocomial infections by antibiotic-resistant Acinetobacter baumannii and the paucity of effective treatment options for such infections, interest in the virulence factors involved in its successful dissemination and propagation in the human host have escalated in recent years. Acinetobacin, a siderophore of A. baumannii, is responsible for iron acquisition under nutritional depravation and has been shown to be one of the key virulence factors for this bacterium. In this Highlight, recent findings regarding various chemical and biological aspects of acinetobactin metabolism closely related to the fitness of A. baumannii at the infection sites have been described. In addition, several notable efforts for identifying novel anti-infectious agents based thereon have been discussed.

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.

MG53-IRS-1 (Mitsugumin 53-Insulin Receptor Substrate-1) Interaction Disruptor Sensitizes Insulin Signaling in Skeletal Muscle.

Mitsugumin 53 (MG53) is an E3 ligase that interacts with and ubiquitinates insulin receptor substrate-1 (IRS-1) in skeletal muscle; thus, an MG53-IRS-1 interaction disruptor (MID), which potentially sensitizes insulin signaling with an elevated level of IRS-1 in skeletal muscle, is an excellent candidate for treating insulin resistance. To screen for an MID, we developed a bimolecular luminescence complementation system using an N-terminal luciferase fragment fused with IRS-1 and a C-terminal luciferase fragment fused with an MG53 C14A mutant that binds to IRS-1 but does not have E3 ligase activity. An MID, which was discovered using the bimolecular luminescence complementation system, disrupted the molecular association of MG53 with IRS-1, thus abolishing MG53-mediated IRS-1 ubiquitination and degradation. Thus, the MID sensitized insulin signaling and increased insulin-elicited glucose uptake with an elevated level of IRS-1 in C2C12 myotubes. These data indicate that this MID holds promise as a drug candidate for treating insulin resistance.

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.

Structural Revision of Baulamycin A and Structure-Activity Relationships of Baulamycin A Derivatives.

Total synthesis of the proposed structure of baulamycin A was performed. The spectral properties of the synthetic compound differ from those reported for the natural product. On the basis of comprehensive NMR study, we proposed two other possible structures for natural baulamycin A. Total syntheses of these two substances were performed, which enabled assignment of the correct structure of baulamycin A. Key features of the convergent and fully stereocontrolled route include Evans Aldol and Brown allylation reactions to construct the left fragment, a prolinol amide-derived alkylation/desymmetrization to install the methyl-substituted centers in the right fragment, and finally, a Carreira alkynylation to join both fragments. In addition, we have determined the inhibitory activities of novel baulamycin A derivatives against the enzyme SbnE. This SAR study provides useful insight into the design of novel SbnE inhibitors that overcome the drug resistance of pathogens, which cause life-threatening infections.

Performance Evaluation of the FREND Cardiac Triple Cartridge on the FREND System.

BACKGROUND: We evaluated the performance of the FREND Cardiac Triple cartridge on the FREND system in the detection of cardiac markers-myoglobin, cardiac troponin I (cTnI), and creatine kinase-MB (CK-MB). METHODS: Quantitative immunoassays were performed using the FREND system (NanoEnTek, Seoul, Korea) and its cartridge. The precision, detection limits, linearity, and correlation with the Siemens Dimension Vista 500 (Siemens Healthcare Diagnostics, Deerfield, IL, USA) were evaluated. The cutoff value for each marker was calculated in healthy individuals (men and women, n = 138 each). RESULTS: The coefficients of variation for imprecision were less than 19.0% at low and high serum concentrations. The lower limits of quantification for myoglobin, cTnI, and CK-MB were 3.11, 0.073, and 0.70 ng/mL, respectively. Acceptable linearity was achieved for each marker (R2 < 0.99). The results from the FREND system were in good agreement with those from the Siemens Dimension Vista (correlation coefficients > 0.9). The cutoff values in male and female individuals (n = 138 each) were 104.3 and 98.9 ng/mL, respectively, for myoglobin, and 4.35 and 5.37 ng/mL, respectively, for CK-MB. The cutoff value for cTnI was 0.073 ng/mL. CONCLUSIONS: The FREND Cardiac Triple cartridge exhibited good precision, clinically acceptable linearity, and reliable correlation with the Dimension Vista.

Development of a novel fluorescence probe capable of assessing the cytoplasmic entry of siderophore-based conjugates.

A novel fluorescence probe capable of assessing the cytoplasmic entry of siderophore-based conjugates was synthesized and evaluated by photochemical characterization and cell-based assays. The specific responsiveness to the cytoplasmic entry of the probe was implemented by adopting a disulfide linker, whose cleavage under the reducing conditions of the cytoplasm induced the display of a distinctive fluorescence signal.

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).