Rational design of a cyclohexanone dehydrogenase for enhanced α,ß-desaturation and substrate specificity.

The selective α,ß-desaturation of cyclic carbonyl compounds, which are found in the core of many steroid and bioactive molecules, using green chemistry is highly desirable. To achieve this task, we have for the first time described and solved the de novo structure of a member of the cyclohexanone dehydrogenase class of enzymes. The breadth of substrate specificity was investigated by assaying the cyclohexanone dehydrogenase, from Alicycliphilus denitrificans, against several cyclic ketones, lactones and lactams. To investigate substrate binding, a catalytic variant, Y195F, was generated and used to obtain a crystallographic complex with the natural substrate, cyclohexanone. This revealed substrate-active site interactions, as well as the proximity of the cofactor, flavin adenine dinucleotide, and enabled us to propose a mechanistic function to key amino acids. We then used molecular dynamic simulations to guide design to add functionality to the cyclohexanone dehydrogenase enzyme. The resulting W113A variant had overall improved enzyme activity and substrate scope, i.e., accepting the bulkier carbonyl compound, dihydrocoumarin. Structural analysis of the W113A variant revealed a broader, more open active site, which helped explain the modified substrate specificity. This work paves the way for future bespoke regioselective α,ß-desaturation in the synthesis of important bioactive molecules via rational enzyme engineering.

Remarkably Selective Binding, Behavior Modification, and Switchable Release of (Bipyridine)3Ru(II) vis-à-vis (Phenanthroline)3Ru(II) by Trimeric Cyclophanes in Water.

A recurring dream of molecular recognition is to create receptors that distinguish between closely related targets with sufficient accuracy, especially in water. The more useful the targets, the more valuable the dream becomes. We now present multianionic trimeric cyclophane receptors with a remarkable ability to bind the iconic (bipyridine)3Ru(II) (with its huge range of applications) while rejecting the nearly equally iconic (phenanthroline)3Ru(II). These receptors not only selectively capture (bipyridine)3Ru(II) but also can be redox-switched to release the guest. 1D- and 2D(ROESY)-NMR spectroscopy, luminescence spectroscopy, and molecular modeling enabled this discovery. This outcome allows the control of these applications, e.g., as a photocatalyst or as a luminescent sensor, by selectively hiding or exposing (bipyridine)3Ru(II). Overall, a 3D nanometric object is selected, picked-up, and dropped-off by a discrete molecular host. The multianionic receptors protect excited states of these metal complexes from phenolate quenchers so that the initial step in photocatalytic phenolate oxidation is retarded by nearly 2 orders of magnitude. This work opens the way for (bipyridine)3Ru(II) to be manipulated in the presence of other functional nano-objects so that many of its applications can be commanded and controlled. We have a cyclophane-based toolkit that can emulate some aspects of proteins that selectively participate in cell signaling and metabolic pathways by changing shape upon environmental commands being received at a location remote from the active site.

Substrate Conformation Regulates Aromatic C-H Vs C-F Bond Activation in Heme-Dependent Tyrosine Hydroxylase.

A recently discovered heme-dependent enzyme tyrosine hydroxylase (TyrH) offers a green approach for functionalizing the high-strength C-H and C-F bonds in aromatic compounds. However, there is ambiguity regarding the nature of the oxidant (compound 0 or compound I) involved in activating these bonds. Herein, using comprehensive molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanical calculations, we reveal that it is compound I (Cpd I) that acts as the primary oxidant involved in the functionalization of both C-F and C-H bonds. The energy barrier for C-H and C-F activation using compound 0 (Cpd 0) as an oxidant was very high, indicating that Cpd 0 cannot be an oxidant. Consistent with the previous experimental finding, our simulation shows two different conformations of the substrate, where one orientation favors the C-H activation, while the other conformation prefers the C-F activation. As such, our mechanistic study shows that nature utilizes just one oxidant, that is, Cpd I, but it is the active site conformation that decides whether it selects C-F or C-H functionalization which may resemble involvement of two different oxidants.

Impact of the T296S mutation in P450 GcoA for aryl-O-demethylation: a QM/MM study.

Lignin, a complex plant cell wall component, holds promise as a renewable aromatic carbon feedstock. p-Vanillin is a key product of lignin depolymerization and a precursor of protocatechuic acid (PCA) that has tremendous potential for biofuel production. While the GcoAB enzyme, native to Amycolatopsis sp., naturally catalyzes aryl-O-demethylation toward guaiacol, recent research introduced a single mutation, T296S, into the GcoAP450 enzyme, enabling it to catalyze aryl-O-demethylation of p-vanillin. This structural modification increases the efficiency of GcoAP450 for the natural substrate while being active for p-vanillin. This study reveals the increased flexibility of p-vanillin and its ability to adapt a favorable conformation by aligning the methoxy group in close proximity to Fe(IV) = O of Cpd I in the active site of the T296S variant. The QM/MM calculations in accordance with the experimental data validated that the rate-limiting step for the oxidation of p-vanillin is hydrogen atom abstraction and provided a detailed geometric structure of stationary and saddle points for the oxidation of p-vanillin.

Single-Site Mutation Induces Water-Mediated Promiscuity in Lignin Breaking Cytochrome P450GcoA.

Cytochrome P450GcoA is an enzyme that catalyzes the guaiacol unit of lignin during the lignin breakdown via an aryl-O-demethylation reaction. This reaction is intriguing and is of commercial importance for its potential applications in the production of biofuel and plastic from biomass feedstock. Recently, the F169A mutation in P450GcoA elicits a promiscuous activity for syringol while maintaining the native activity for guaiacol. Using comprehensive MD simulations and hybrid QM/MM calculations, we address, herein, the origin of promiscuity in P450GcoA and its relevance to the specific activity toward lignin-derived substrates. Our study shows a crucial role of an aromatic dyad of F169 and F395 by regulating the water access to the catalytic center. The F169A mutation opens a water aqueduct and hence increases the native activity for G-lignin. We show that syringol binds very tightly to the WT enzyme, which blocks the conformational rearrangement needed for the second step of O-demethylation. The F169A creates an extra room favoring the conformational rearrangement in the 3-methoxycatechol (3MC) and second dose of the dioxygen insertion. Therefore, using MD simulations and complemented by thorough QM/MM calculations, our study shows how a single-site mutation rearchitects active site engineering for promiscuous syringol activity.

Taming Tris(bipyridine)ruthenium(II) and Its Reactions in Water by Capture/Release with Shape-Switchable Symmetry-Matched Cyclophanes.

Electron/proton transfers in water proceeding from ground/excited states are the elementary reactions of chemistry. These reactions of an iconic class of molecules─polypyridineRu(II)─are now controlled by capturing or releasing three of them with hosts that are shape-switchable. Reversible erection or collapse of the host walls allows such switchability. Some reaction rates are suppressed by factors of up to 120 by inclusive binding of the metal complexes. This puts nanometric coordination chemistry in a box that can be open or shut as necessary. Such second-sphere complexation can allow considerable control to be exerted on photocatalysis, electrocatalysis, and luminescent sensing involving polypyridineRu(II) compounds. The capturing states of hosts are symmetry-matched to guests for selective binding and display submicromolar affinities. A perching complex, which is an intermediate state between capturing and releasing states, is also demonstrated.

Simulations reveal the key role of Arg15 in the promiscuous activity in the HisA enzyme.

The HisA enzyme catalyzes the first step of histidine biosynthesis via the Amadori rearrangement of the substrate ProFAR. Since it possesses the most conserved and ancient TIM-barrel fold, it provides an ideal framework for bioengineering of a new function from ancestral enzymes. In the present study, first, the catalytic mechanism of HisA biosynthesis was elucidated using hybrid Quantum Mechanical/Molecular Mechanical calculations, and thereafter, key residues contributing towards the promiscuity for TrpF activity were revealed using several MD simulations of a wild type enzyme and its variant with the native (ProFAR) and promiscuous (PRA) substrates. Our study reveals that the two loops (ßα)1 and (ßα)5 on the catalytic site of the HisA enzyme have incredible adaptability for the native and promiscuous substrates. The conformational interplay between these two loops is substrate driven and precise bioengineering targeting these loops is key to the emergence of new functions. Furthermore, the study reveals a key role of the Arg 15 residue which is close to the catalytic center of the enzyme in the bifunctionality of the HisA enzyme by increasing the loop flexibility. Therefore, our study provides crucial information for future bioengineering work to use the HisA enzyme as a scaffold for new enzymatic activity.

Thebaine is Selectively Demethylated by Thebaine 6-O-Demethylase and Codeine-3-O-demethylase at Distinct Binding Sites: A Computational Study.

Two homologous 2-oxoglutarate-dependent (ODD) nonheme enzymes thebaine 6-O-demethylase (T6ODM) and codeine-3-O-demethylase (CODM), are involved in the morphine biosynthesis pathway from thebaine, catalyzing the O-demethylation reaction with precise regioselectivity at C6 and C3 positions of thebaine respectively. We investigated the origin of the regioselectivity of these enzymes by combined molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations and found that Thebaine binds at the two distinct sites of T6ODM and CODM, which determines the regioselectivity of the enzymes. A remarkable oxo rotation is observed in the decarboxylation process. Starting from the closed pentacoordinate configuration, the C-terminal lid adopts an open conformation in the octahedral Fe(IV) = O complex to facilitate the subsequent demethylation. Phe241 and Phe311 stabilize the substrate in the binding pocket, while Arg219 acts as a gatekeeper residue to stabilize the substrate. Our results unravel the regioselectivity in 2-OG dependent nonheme enzymes and may shed light for exploring the substrate scope of these enzymes and developing novel biotechnology for morphine biosynthesis.

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis.

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.

Regio- and stereoselectivity in the CYP450BM3-catalyzed hydroxylation of complex terpenoids: a QM/MM study.

The site-selective C-H oxidation of terpenoids by P450 attracts great attention because of their wide range of biological activities. However, the binding and catalytic mechanism of P450 for the hydroxylation of complex terpenoid substrates remains elusive, which has limited the rational engineering of P450 as a biocatalyst for terpenoid biosynthesis. Here, we studied the origin of the selectivity and reactivity of P450BM3 in the hydroxylation of terpenoids by combining molecular dynamics simulations and QM/MM calculations, using artemisinin as a model compound. We found that the conformational change of the ß1 sheet at the substrate entrance and the displacement of the ß' helix were critical for reshaping the binding pocket to modulate substrate entrance and positioning the C-H to be activated toward the oxidative species of P450 for the subsequent hydrogen abstraction, the rate-determining step of hydroxylation. There is a distinct linear correlation between activation barriers and reaction coordinates, indicating that reaction coordinates can be used as a facile descriptor for predicting the reactivity of P450BM3. These findings would provide valuable guidance for predicting the selectivity and reactivity of P450BM3 for the selective hydroxylation of non-native terpenoid substrates so as to prioritize the rationally designed enzymes for terpenoid biosynthesis.

The complex structure of bile salt hydrolase from Lactobacillus salivarius reveals the structural basis of substrate specificity.

The gut bacterial bile salt hydrolase (BSH) plays a critical role in host lipid metabolism and energy harvest. Therefore, BSH is a promising microbiome target to develop new therapies to regulate obesity in humans and novel non-antibiotic growth promoters for food animals. We previously reported the 1.90 Å apo crystal structure of BSH from Lactobacillus salivarius (lsBSH). In this study, we soaked the lsBSH crystal with glycocholic acid (GCA), a substrate, and obtained a 2.10 Å structure containing complex of lsBSH bound to GCA and cholic acid (CA), a product. The substrate/product sits in the water-exposed cavity molded by Loops 2 and 3. While the glycine moiety of GCA is exposed into a highly polar pocket, the sterane core of GCA is stabilized by aromatic and hydrophobic interactions. Comparison of product binding with BSH from Clostridium perfringenes reveals a distinct orientation of the sterane core in the binding site. The stability of the substrate-lsBSH complex and the putative catalytic mechanism were explored with molecular dynamics simulations. Site-directed mutagenesis of lsBSH demonstrated that Cys2 and Asn171 are critical for enzymatic activity, while Tyr24, Phe65 and Gln257 contribute to the substrate specificity. Together, this study provides structural insights into BSH-substrate interaction, the mechanism of catalysis and substrate specificity, which facilitate rational design of BSH inhibitors.

Reaction Mechanism of Histone Demethylation in αKG-dependent Non-Heme Iron Enzymes.

Histone demethylases (KDMs) catalyze histone lysine demethylation, an important epigenetic process that controls gene expression in eukaryotes, and represent important cancer drug targets for cancer treatment. Demethylation of histone is comprised of sequential reaction steps including oxygen activation, decarboxylation, and demethylation. The initial oxygen binding and activation steps have been studied. However, the information on the complete catalytic reaction cycle is limited, which has impeded the structure-based design of inhibitors targeting KDMs. Here we report the mechanism of the complete reaction steps catalyzed by a representative nonheme iron αKG-dependent KDM, PHF8 using QM/MM approaches. The atomic-level understanding on the complete reaction mechanism of PHF8 would shed light on the structure-based design of selective inhibitors targeting KDMs to intervene in cancer epigenetics.

Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105-/His224+ rather than a concerted process.

Mechanism of Phosphatidylglycerol Activation Catalyzed by Prolipoprotein Diacylglyceryl Transferase.

Lipoproteins are essential for bacterial survival. Bacterial lipoprotein biosynthesis is accomplished by sequential modification by three enzymes in the inner membrane, all of which are emerging antimicrobial targets. The X-ray crystal structure of prolipoprotein diacylglyceryl transferase (Lgt) and apolipoprotein N-acyl transferase (Lnt) has been reported. However, the mechanisms of the post-translational modification catalyzed by these enzymes have not been understood. Here, we studied the mechanism of the transacylation reaction catalyzed by Lgt, the first enzyme for lipoprotein modification using molecular docking, molecular dynamics, and quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that Arg143, Arg239, and Glu202 play a critical role in stabilizing the glycerol-1-phosphate head group and activating the glycerol C3-O ester bond of the phosphatidylglycerol (PG) substrate. With PG binding, the opening of the L6-7 loop mediated by the highly conserved Arg236 residue as a gatekeeper is observed, which facilitates the release of the modified lipoprotein product, as well as the entry of another PG substrate. Further QM/MM studies revealed that His103 acts as a catalytic base to abstract a proton from the cysteine residue of the preproliprotein, initiating the diacylglyceryl transfer from PG to preprolipoprotein. This is the first study on the mechanism of lipoprotein modification catalyzed by a post-translocational processing enzyme. The transacylation mechanism of Lgt would shed light on the development of novel antimicrobial therapies targeting the challenging enzymes involved in the post-translocational modification pathway of lipoproteins.

Stereodivergent Protein Engineering of a Lipase To Access All Possible Stereoisomers of Chiral Esters with Two Stereocenters.

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants of each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultrasmall mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.

Reaction Mechanism of Isopentenyl Phosphate Kinase: A QM/MM Study.

Isopentenyl phosphate kinase (IPK) catalyzes the Mg2+-ATP dependent phosphorylation reactions to produce isopentenyl diphosphate, an important precursor in the synthesis of isopentenols. However, the position of the divalent metal ion in the crystal structures of IPK in complex with ATP and its native substrate IP has not been definitively resolved, and as a result ambiguity surrounds the catalytic mechanism of IP, limiting its exploitation as a biofuel and in drug design. Here we report the catalytically competent structure in complex with the metal ion Mg2+ and elucidate the phosphorylation reaction mechanism using molecular dynamic simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B97d/AMBER99). Comparing the substrate-bound and substrate-free IPK complexes, we observed that substrate binding results in significant conformational change of three residues Lys204, Glu207, and Lys211 located on the αG helix to form a strong salt bridge network with Asp145, which in turn tethers the invariant Ser142 via H-bond interaction. The conformational change shuts the subtrate entrance channel formed between the αG and αE helices. Further, we demonstrate the phosphorylation reaction occurs with a reaction barrier of 17.58 kcal/mol, which is in agreement with the previous experimental kinetic data. We found that a highly conserved Gly8 on a glycine-rich loop, together with Lys14, stabilizes the transition state.

Effects of Mutations on Structure-Function Relationships of Matrix Metalloproteinase-1.

Matrix metalloproteinase-1 (MMP-1) is one of the most widely studied enzymes involved in collagen degradation. Mutations of specific residues in the MMP-1 hemopexin-like (HPX) domain have been shown to modulate activity of the MMP-1 catalytic (CAT) domain. In order to reveal the structural and conformational effects of such mutations, a molecular dynamics (MD) study was performed of in silico mutated residues in the X-ray crystallographic structure of MMP-1 complexed with a collagen-model triple-helical peptide (THP). The results indicate an important role of the mutated residues in MMP-1 interactions with the THP and communication between the CAT and the HPX domains. Each mutation has a distinct impact on the correlated motions in the MMP-1•THP. An increased collagenase activity corresponded to the appearance of a unique anti-correlated motion and decreased correlated motions, while decreased collagenase activity corresponded both to increased and decreased anti-correlated motions.

Importance of the Linker Region in Matrix Metalloproteinase-1 Domain Interactions.

Collagenolysis is catalyzed by enzymes from the matrix metalloproteinase (MMP) family, where one of the most studied is MMP-1. The X-ray crystallographic structure of MMP-1 complexed with a collagen-model triple-helical peptide (THP) provided important atomistic information, but few details on the effects of the conformational flexibility on catalysis. In addition, the role of the linker region between the catalytic (CAT) and hemopexin-like (HPX) domains was not defined. In order to reveal the dynamics and correlations of MMP-1 comprehensive atomistic molecular dynamics simulations of an MMP-1•THP complex was performed. To examine the role of the linker region for MMP-1 function simulations with linker regions from MT1-MMP/MMP-14 and MMP-13 replacing the MMP-1 linker region were performed. The MD studies were in good agreement with the experimental observation that in the MMP-1•THP X-ray crystallographic structure MMP-1 is in a "closed" conformation. MD revealed that the interactions of the THP with the both the CAT and HPX domains of MMP-1 are dynamic in nature, and the linker region of MMP-1 influences the interactions and dynamics of both the CAT and HPX domains and collagen binding to MMP-1.

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase.

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.