Directed Evolution of Protoglobin Optimizes the Enzyme Electric Field.

To unravel why computational design fails in creating viable enzymes, while directed evolution (DE) succeeds, our research delves into the laboratory evolution of protoglobin. DE has adapted this protein to efficiently catalyze carbene transfer reactions. We show that the previously proposed enhanced substrate access and binding alone cannot account for increased yields during DE. The 3D electric field in the entire active site is tracked through protein dynamics, clustered using the affinity propagation algorithm, and subjected to principal component analysis. This analysis reveals notable changes in the electric field with DE, where distinct field topologies influence transition state energetics and mechanism. A chemically meaningful field component emerges and takes the lead during DE and facilitates crossing the barrier to carbene transfer. Our findings underscore intrinsic electric field dynamic's influence on enzyme function, the ability of the field to switch mechanisms within the same protein, and the crucial role of the field in enzyme design.

The Unique Role of the Second Coordination Sphere to Unlock and Control Catalysis in Nonheme Fe(II)/2-Oxoglutarate Histone Demethylase KDM2A.

Nonheme Fe(II) and 2-oxoglutarate (2OG)-dependent histone lysine demethylases 2A (KDM2A) catalyze the demethylation of the mono- or dimethylated lysine 36 residue in the histone H3 peptide (H3K36me1/me2), which plays a crucial role in epigenetic regulation and can be involved in many cancers. Although the overall catalytic mechanism of KDMs has been studied, how KDM2 catalysis takes place in contrast to other KDMs remains unknown. Understanding such differences is vital for enzyme redesign and can help in enzyme-selective drug design. Herein, we employed molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) to explore the complete catalytic mechanism of KDM2A, including dioxygen diffusion and binding, dioxygen activation, and substrate oxidation. Our study demonstrates that the catalysis of KDM2A is controlled by the conformational change of the second coordination sphere (SCS), specifically by a change in the orientation of Y222, which unlocks the 2OG rearrangement from off-line to in-line mode. The study demonstrates that the variant Y222A makes the 2OG rearrangement more favorable. Furthermore, the study reveals that it is the size of H3K36me3 that prevents the 2OG rearrangement, thus rendering the enzyme inactivity with trimethylated lysine. Calculations show that the SCS and long-range interacting residues that stabilize the HAT transition state in KDM2A differ from those in KDM4A, KDM7B, and KDM6A, thus providing the basics for the enzyme-selective redesign and modulation of KDM2A without influencing other KDMs.

From random to rational: improving enzyme design through electric fields, second coordination sphere interactions, and conformational dynamics.

Enzymes are versatile and efficient biological catalysts that drive numerous cellular processes, motivating the development of enzyme design approaches to tailor catalysts for diverse applications. In this perspective, we investigate the unique properties of natural, evolved, and designed enzymes, recognizing their strengths and shortcomings. We highlight the challenges and limitations of current enzyme design protocols, with a particular focus on their limited consideration of long-range electrostatic and dynamic effects. We then delve deeper into the impact of the protein environment on enzyme catalysis and explore the roles of preorganized electric fields, second coordination sphere interactions, and protein dynamics for enzyme function. Furthermore, we present several case studies illustrating successful enzyme-design efforts incorporating enzyme strategies mentioned above to achieve improved catalytic properties. Finally, we envision the future of enzyme design research, spotlighting the challenges yet to be overcome and the synergy of intrinsic electric fields, second coordination sphere interactions, and conformational dynamics to push the state-of-the-art boundaries.

Catalysis by KDM6 Histone Demethylases - A Synergy between the Non-Heme Iron(II) Center, Second Coordination Sphere, and Long-Range Interactions.

KDM6A (UTX) and KDM6B (JMJD3) are human non-heme Fe(II) and 2-oxoglutarate (2OG) dependent JmjC oxygenases that catalyze the demethylation of trimethylated lysine 27 in the N-terminal tail of histone H3, a post-translational modification that regulates transcription. A Combined Quantum Mechanics/ Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) study on the catalytic mechanism of KDM6A/B reveals that the transition state for the rate-limiting hydrogen atom transfer (HAT) reaction in KDM6A catalysis is stabilized by polar (Asn217) and aromatic (Trp369)/non-polar (Pro274) residues in contrast to KDM4, KDM6B and KDM7 demethylases where charged residues (Glu, Arg, Asp) are involved. KDM6A employs both σ- and π-electron transfer pathways for HAT, whereas KDM6B employs the σ-electron pathway. Differences in hydrogen bonding of the Fe-chelating Glu252(KDM6B) contribute to the lower energy barriers in KDM6B vs. KDM6A. The study reveals a dependence of the activation barrier of the rebound hydroxylation on the Fe-O-C angle in the transition state of KDM6A. Anti-correlation of the Zn-binding domain with the active site residues is a key factor distinguishing KDM6A/B from KDM7/4s. The results reveal the importance of communication between the Fe center, second coordination sphere, and long-range interactions in catalysis by KDMs and, by implication, other 2OG oxygenases.

Can an external electric field switch between ethylene formation and L-arginine hydroxylation in the ethylene forming enzyme?

The non-heme Fe(II) and 2-oxoglutarate (2OG) dependent ethylene-forming enzyme (EFE) catalyzes both ethylene generation and L-Arg hydroxylation. Despite experimental and computational progress in understanding the mechanism of EFE, no EFE variant has been optimized for ethylene production while simultaneously reducing the L-Arg hydroxylation activity. In this study, we show that the two L-Arg binding conformations, associated with different reactivity preferences in EFE, lead to differences in the intrinsic electric field (IntEF) of EFE. Importantly, we suggest that applying an external electric field (ExtEF) along the Fe-O bond in the EFE·Fe(III)·OO-˙·2OG·L-Arg complex can switch the EFE reactivity between L-Arg hydroxylation and ethylene generation. Furthermore, we explored how applying an ExtEF alters the geometry, electronic structure of the key reaction intermediates, and the individual energy contributions of second coordination sphere (SCS) residues through combined quantum mechanics/molecular mechanics (QM/MM) calculations. Experimentally generated variant forms of EFE with alanine substituted for SCS residues responsible for stabilizing the key intermediates in the two reactions of EFE led to changes in enzyme activity, thus demonstrating the key role of these residues. Overall, the results of applying an ExtEF indicate that making the IntEF of EFE less negative and stabilizing the off-line binding of 2OG is predicted to increase ethylene generation while reducing L-Arg hydroxylation.

Dioxygen Binding Is Controlled by the Protein Environment in Non-heme FeII and 2-Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene-Forming Enzyme.

Invited for the cover of this issue are Christo Z. Christov and co-workers at Michigan Technological University, University of Oxford, and Michigan State University. The image depicts the oxygen diffusion channel in class 7 histone demethylase (PHF8) and ethylene-forming enzyme (EFE) and changes in the enzymes' conformations upon binding. Read the full text of the article at 10.1002/chem.202300138.

Dioxygen Binding Is Controlled by the Protein Environment in Non-heme FeII and 2-Oxoglutarate Oxygenases: A Study on Histone Demethylase PHF8 and an Ethylene-Forming Enzyme.

This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme FeII /2OG enzymes: a class 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene-forming enzyme (EFE), which catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially bind 2OG by using an off-line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in-line mode, whereas EFE is catalytically productive for ethylene production from 2OG in the off-line mode. We used classical molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics (QM/MM-MetD) simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in-line FeIII -OO⋅- intermediate in PHF8 and the off-line FeIII -OO⋅- intermediate in EFE.

How Human TET2 Enzyme Catalyzes the Oxidation of Unnatural Cytosine Modifications in Double-Stranded DNA.

Methylation of cytosine bases is strongly linked to gene expression, imprinting, aging, and carcinogenesis. The Ten-eleven translocation (TET) family of enzymes, which are Fe(II)/2-oxoglutarate (2OG)-dependent enzymes, employ Fe(IV)=O species to dealkylate the lesioned bases to an unmodified cytosine. Recently, it has been shown that the TET2 enzyme can catalyze promiscuously DNA substrates containing unnatural alkylated cytosine. Such unnatural substrates of TET can be used as direct probes for measuring the TET activity or capturing TET from cellular samples. Herein, we studied the catalytic mechanisms during the oxidation of the unnatural C5-position modifications (5-ethylcytosine (5eC), 5-vinylcytosine (5vC) and 5-ethynylcytosine (5eyC)) and the demethylation of N4-methylated lesions (4-methylcytosine (4mC) and 4,4-dimethylcytosine(4dmC)) of the cytosine base by the TET2 enzyme using molecular dynamics (MD) and combined quantum mechanics and molecular mechanics (QM/MM) computational approaches. The results reveal that the chemical nature of the alkylation of the double-stranded (ds) DNA substrates induces distinct changes in the interactions in the binding site, the second coordination sphere, and long-range correlated motions of the ES complexes. The rate-determining hydrogen atom transfer (HAT) is faster in N4-methyl substituent substrates than in the C5-alkylations. Importantly, the calculations show the preference of hydroxylation over desaturation in both 5eC and 5vC substrates. The studies elucidate the post-hydroxylation rearrangements of the hydroxylated intermediates of 5eyC and 5vC to ketene and 5-formylmethylcytosine (5fmC), respectively, and hydrolysis of hemiaminal intermediate of 4mC to formaldehyde and unmodified cytosine proceed exclusively in aqueous solution outside of the enzyme environment. Overall, the studies show that the chemical nature of the unnatural alkylated cytosine substrates exercises distinct effects on the binding interactions, reaction mechanism, and dynamics of TET2.

Can Second Coordination Sphere and Long-Range Interactions Modulate Hydrogen Atom Transfer in a Non-Heme Fe(II)-Dependent Histone Demethylase?

Fe(II)-dependent oxygenases employ hydrogen atom transfer (HAT) to produce a myriad of products. Understanding how such enzymes use dynamic processes beyond the immediate vicinity of the active site to control the selectivity and efficiency of HAT is important for metalloenzyme engineering; however, obtaining such knowledge by experiments is challenging. This study develops a computational framework for identifying second coordination sphere (SCS) and especially long-range (LR) residues relevant for catalysis through dynamic cross-correlation analysis (DCCA) using the human histone demethylase PHF8 (KDM7B) as a model oxygenase. Furthermore, the study explores the mechanistic pathways of influence of the SCS and LR residues on the HAT reaction. To demonstrate the plausibility of the approach, we investigated the effect of a PHF8 F279S clinical mutation associated with X-linked intellectual disability, which has been experimentally shown to ablate PHF8-catalyzed demethylation. In agreement, the molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies showed a change in the H31-14K9me2 substrate orientation and an increased HAT barrier. We systematically analyzed the pathways by which the identified SCS and LR residues may influence HAT by exploring changes in H3K9me2 substrate orientation, interdomain correlated motions, HAT transition state stabilization, reaction energetics, electron transfer mechanism, and alterations in the intrinsic electric field of PHF8. Importantly, SCS and LR variations decrease key motions of α9-α12 of the JmjC domain toward the Fe(IV)-center that are associated with tighter binding of the H31-14K9me2 substrate. SCS and LR residues alter the intrinsic electric field of the enzyme along the reaction coordinate and change the individual energetic contributions of residues toward TS stabilization. The overall results suggest that DCCA can indeed identify non-active-site residues relevant for catalysis. The substitutions of such dynamically correlated residues might be used as a tool to tune HAT in non-heme Fe(II)- and 2OG-dependent enzymes.

A synergy between the catalytic and structural Zn(II) ions and the enzyme and substrate dynamics underlies the structure-function relationships of matrix metalloproteinase collagenolysis.

Matrix metalloproteinases (MMPs) are Zn(II) dependent endopeptidases involved in the degradation of collagen. Unbalanced collagen breakdown results in numerous pathological conditions, including cardiovascular and neurodegenerative diseases and tumor growth and invasion. Matrix metalloproteinase-1 (MMP-1) is a member of the MMPs family. The enzyme contains catalytic and structural Zn(II) ions. Despite many studies on the enzyme, there is little known about the synergy between the two Zn(II) metal ions and the enzyme and substrate dynamics in MMP-1 structure-function relationships. We performed a computational study of the MMP-1•triple-helical peptide (THP) enzyme•substrate complex to provide this missing insight. Our results revealed Zn(II) ions' importance in modulating the long-range correlated motions in the MMP-1•THP complex. Overall, our results reveal the importance of the catalytic Zn(II) and the role of the structural Zn(II) ion in preserving the integrity of the enzyme active site and the overall enzyme-substrate complex synergy with the dynamics of the enzyme and the substrate. Notably, both Zn(II) sites participate in diverse networks of long-range correlated motions that involve the CAT and HPX domains and the THP substrate, thus exercising a complex role in the stability and functionality of the MMP-1•THP complex. Both the Zn(II) ions have a distinct impact on the structural stability and dynamics of the MMP-1•THP complex. The study shifts the paradigm from the "local role" of the Zn(II) ions with knowledge about their essential role in the long-range dynamics and stability of the overall enzyme•substrate (ES) complex.

Effects of the Nature of the Metal Ion, Protein and Substrate on the Catalytic Center in Matrix Metalloproteinase-1: Insights from Multilevel MD, QM/MM and QM Studies.

Matrix metalloproteinase-1 (MMP-1) is a Zn(II) dependent endopeptidase involved in the degradation of collagen, the most abundant structural protein in the extracellular matrix of connective tissues and the human body. Herein we performed a multilevel computational analysis including molecular dynamics (MD), combined quantum mechanics/molecular mechanics (QM/MM), and quantum mechanics (QM) calculations to characterize the structure and geometry of the catalytic Zn(II) within the MMP-1 protein environment in comparison to crystallographic and spectroscopic data. The substrate's removal fine-tuned impact on the conformational dynamics and geometry of the catalytic Zn(II) center was also explored. Finally, the study examined the effect of substituting catalytic Zn(II) by Co(II) on the overall structure and dynamics of the MMP-1 THP complex and specifically on the geometry of the catalytic metal center. Overall our QM/MM and QM studies were in good agreement with the MM description of the Zn(II) centers in the MD simulations.

Role of Structural Dynamics in Selectivity and Mechanism of Non-heme Fe(II) and 2-Oxoglutarate-Dependent Oxygenases Involved in DNA Repair.

AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (m3C) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic ß-hairpin, are involved in substrate binding in AlkBH2-dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)=O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2-dsDNA and AlkB-dsDNA; in AlkB-ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein-DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism.

Catalysis by the Non-Heme Iron(II) Histone Demethylase PHF8 Involves Iron Center Rearrangement and Conformational Modulation of Substrate Orientation.

PHF8 (KDM7B) is a human non-heme 2-oxoglutarate (2OG) JmjC domain oxygenase that catalyzes the demethylation of the di/mono-Nε-methylated K9 residue of histone H3. Altered PHF8 activity is linked to genetic diseases and cancer; thus, it is an interesting target for epigenetic modulation. We describe the use of combined quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of PHF8, including dioxygen activation, 2OG binding modes, and substrate demethylation steps. A PHF8 crystal structure manifests the 2OG C-1 carboxylate bound to iron in a nonproductive orientation, i.e., trans to His247. A ferryl-oxo intermediate formed by activating dioxygen bound to the vacant site in this complex would be nonproductive, i.e., "off-line" with respect to reaction with Nε-methylated K9. We show rearrangement of the "off-line" ferryl-oxo intermediate to a productive "in-line" geometry via a solvent exchange reaction (called "ferryl-flip") is energetically unfavorable. The calculations imply that movement of the 2OG C-1 carboxylate prior to dioxygen binding at a five-coordination stage in catalysis proceeds with a low barrier, suggesting that two possible 2OG C-1 carboxylate geometries can coexist at room temperature. We explored alternative mechanisms for hydrogen atom transfer and show that second sphere interactions orient the Nε-methylated lysine in a conformation where hydrogen abstraction from a methyl C-H bond is energetically more favorable than hydrogen abstraction from the N-H bond of the protonated Nε-methyl group. Using multiple HAT reaction path calculations, we demonstrate the crucial role of conformational flexibility in effective hydrogen transfer. Subsequent hydroxylation occurs through a rebound mechanism, which is energetically preferred compared to desaturation, due to second sphere interactions. The overall mechanistic insights reveal the crucial role of iron-center rearrangement, second sphere interactions, and conformational flexibility in PHF8 catalysis and provide knowledge useful for the design of mechanism-based PHF8 inhibitors.

Catalysis by the JmjC histone demethylase KDM4A integrates substrate dynamics, correlated motions and molecular orbital control.

The N ε-methyl lysine status of histones is important in the regulation of eukaryotic transcription. The Fe(ii) and 2-oxoglutarate (2OG) -dependent JmjC domain enzymes are the largest family of histone N ε-methyl lysine demethylases (KDMs). The human KDM4 subfamily of JmjC KDMs is linked with multiple cancers and some of its members are medicinal chemistry targets. We describe the use of combined molecular dynamics (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) methods to study the mechanism of KDM4A, which catalyzes demethylation of both tri- and di-methylated forms of histone H3 at K9 and K36. The results show that the oxygen activation at the active site of KDM4A is optimized towards the generation of the reactive Fe(iv)-oxo intermediate. Factors including the substrate binding mode, correlated motions of the protein and histone substrates, and molecular orbital control synergistically contribute to the reactivity of the Fe(iv)-oxo intermediate. In silico substitutions were performed to investigate the roles of residues (Lys241, Tyr177, and Asn290) in substrate orientation. The Lys241Ala substitution abolishes activity due to altered substrate orientation consistent with reported experimental studies. Calculations with a macrocyclic peptide substrate analogue reveal that induced conformational changes/correlated motions in KDM4A are sequence-specific in a manner that influences substrate binding affinity. Second sphere residues, such as Ser288 and Thr289, may contribute to KDM4A catalysis by correlated motions with active site residues. Residues that stabilize key intermediates, and which are predicted to be involved in correlated motions with other residues in the second sphere and beyond, are shown to be different in KDM4A compared to those in another JmjC KDM (PHF8), which acts on H3K9 di- and mono-methylated forms, suggesting that allosteric type inhibition is of interest from the perspective of developing selective JmjC KDM inhibitors.

Structure-function relationships in KDM7 histone demethylases.

The demethylation of lysine residues of histone proteins is a key epigenetic mechanism in cells. The enzymes that catalyze these processes are called histone demethylases (KDMs). The largest family of KDMs is the Jumonji C (JmjC) domain-containing enzymes; these includes KDM2-7 subfamily of enzymes. The JmjC proteins are Fe(II) and 2-Oxoglutarate (2OG) - dependent dioxygenases that couple substrate oxidation to decarboxylation of 2OG to form succinate and CO2. The KDM7 subfamily of enzymes - PHF8 (KDM7B) and KIAA1718 (KDM7A) are human JmjC 2OG-dependent Nε-methyl lysine demethylases and are involved in demethylation of lysine residues in histones such as H3K27me2/1, H3K9me2/1 and H4K20me1. These enzymes are involved in multiple pathologic processes, including cancers and mental retardation. In this chapter, we present the current state of the art in the structural, biochemical and computational studies of KDM7 enzymes.

Conformational Dynamics Underlies Different Functions of Human KDM7 Histone Demethylases.

The human KDM7 subfamily histone H3 Nϵ-methyl lysine demethylases PHF8 (KDM7B) and KIAA1718 (KDM7A) have different substrate selectivities and are linked to genetic diseases and cancer. We describe experimentally based computational studies revealing that flexibility of the region linking the PHD finger and JmjC domains in PHF8 and KIAA1718 regulates interdomain interactions, the nature of correlated motions, and ultimately H3 binding and demethylation site selectivity. F279S an X-linked mental retardation mutation in PHF8 is involved in correlated motions with the iron ligands and second sphere residues. The calculations reveal key roles of a flexible protein environment in productive formation of enzyme-substrate complexes and suggest targeting the flexible KDM7 linker region is of interest from a medicinal chemistry perspective.

Conformational flexibility influences structure-function relationships in nucleic acid N-methyl demethylases.

N-Methylation of DNA/RNA bases can be regulatory or damaging and is linked to diseases including cancer and genetic disorders. Bacterial AlkB and human FTO are DNA/RNA demethylases belonging to the Fe(ii) and 2-oxoglutarate oxygenase superfamily. Modelling studies reveal conformational dynamics influence structure-function relationships of AlkB and FTO, e.g. why 1-methyladenine is a better substrate for AlkB than 6-methyladenine. Simulations show that the flexibility of the double stranded DNA substrate in AlkB influences correlated motions, including between the core jelly-roll fold and an active site loop involved in substrate binding. The FTO N- and C-terminal domains move in respect to one another in a manner likely important for substrate binding. Substitutions, including clinically observed ones, influencing catalysis contribute to the network of correlated motions in AlkB and FTO. Overall, the calculations highlight the importance of the overall protein environment and its flexibility to the geometry of the reactant complexes.

Integrating molecular probes and molecular dynamics to reveal binding modes of GLUT5 activatory and inhibitory ligands.

Fructose transporter GLUT5 is characterized by unusual substrate specificity and is linked to a variety of metabolic disorders. A series of high-affinity GLUT5-specific sugar-based probes - ManCous - have been very recently described and efficiently used as reporters of GLUT5 activity in cells. Here we present several 1 microsecond molecular dynamics (MD) simulations of GLUT5 and its complexes with fructose and two different ManCou probes, in a solvated cell membrane environment. These simulations show key molecular interactions within GLUT5 that promote the passage of the substrate through vs. blockage of the transport mechanism. Identification of these specific interactions and their long-range effects on GLUT5 structure and dynamics provides an essential basis for the future development of GLUT5-specific inhibitors.