Engineering of Rieske dioxygenase variants with improved cis-dihydroxylation activity for benzoates.

Rieske dioxygenases are enzyme systems that have a long history of being applied as chiral, green chemical catalysts in the production of valuable building blocks for organic synthesis, owing to their ability to catalyze the cis‐dihydroxylation of aromatics. The practical utility of these catalysts, however, has been limited by restrictions on their substrate scope and selectivity. Recent studies have demonstrated the potential of modifying the substrate tunnel of oxidase enzymes to modulate the selectivity and activity of these enzymes for specific substrates. Herein, we report the targeted modification of residues lining the substrate tunnel of a representative and widely used Rieske dioxygenase, toluene dioxygenase (TDO). Several enzyme variants generated through modification of the residues lining the substrate tunnel demonstrated substantially improved activity over the wild‐type enzyme for multiple substrates. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in efforts to elucidate how the identified mutations resulted in improved activity. These analyses suggested that new interactions introduced along the substrate tunnel may explain the improved activity observed with the best‐performing enzyme variants.

Rieske dioxygenases are multi-component enzyme systems, naturally found in many soil bacteria, that have been widely applied in the production of fine chemicals, owing to the unique and valuable oxidative dearomatization reactions they catalyze. The range of practical applications for these enzymes in this context has historically been limited, however, due to their limited substrate scope and strict selectivity. In an attempt to overcome these limitations, our research group has employed the tools of enzyme engineering to expand the substrate scope or improve the reactivity of these enzyme systems in specific contexts. Traditionally, enzyme engineering campaigns targeting metalloenzymes have avoided mutations to metal-coordinating residues, based on the assumption that these residues are essential for enzyme activity. Inspired by the success of other recent enzyme engineering reports, our research group investigated the potential to alter or improve the reactivity of Rieske dioxygenases by altering or eliminating iron coordination in the active site of these enzymes. Herein, we report the modification of all three iron-coordinating residues in the active site of toluene dioxygenase both to alternate residues capable of coordinating iron, and to a residue that would eliminate iron coordination. The enzyme variants produced in this way were tested for their activity in the cis-dihydroxylation of a small library of potential aromatic substrates. The results of these studies demonstrated that all three iron-coordinating residues, in their natural state, are essential for enzyme activity in toluene dioxygenase, as the introduction of any mutations at these sites resulted in a complete loss of cis-dihydroxylation activity.

Rieske dioxygenases have a history of utility in organic synthesis, owing to their ability to catalyze the asymmetric dihydroxylation of aromatics to produce chiral diene‐diol metabolites. However, their utility as green‐chemical tools has been limited by steric and electronic constraints on their substrate scopes and their activity. Herein we report the rational engineering of a widely used Rieske dioxygenase, toluene dioxygenase (TDO), to improve the activity of this enzyme system for the dihydroxylation of a synthetically valuable substrate class for which the wild‐type enzyme possesses low activity, the ester‐functionalized aromatics. Through active site targeted mutagenesis and application of a recently reported high throughput screening platform, engineered TDO variants with significantly increased activity in the dihydroxylation of these valuable substrates were identified and characterized, revealing key active site residues that modulate the enzyme's activity and selectivity.

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase β discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn(2+) for Mg(2+) decreased sugar discrimination for both wild-type and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2' of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 Å, respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase β utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

Resistance to existing drugs of tuberculosis bacteria demands an immediate requirement to develop effective new chemical entities acting on emerging targets. Seryl-tRNA synthetase (SerRS) is essential for the viability of Mycobacterium tuberculosis (MTB). In this study, we have attempted to develop the tertiary structure of SerRS through homology modelling and to elucidate the active site interactions of inhibitor compounds aided by docking. Homology modelling using PDB ID: '2DQ3: A' chain as template and validation of the model was carried out with Modeller V9.13 and SAVES online server respectively. About 1248 compounds from a putative kinase compound library of PubChem database found active in whole cell bioassay (AID2842) on MTB - H37Rv was used in docking studies using 'AutoDock'. Out of the tested molecules, nine showed docking scores ≤-10 kcal/mol with good drug-like properties were further subjected to molecular dynamics (MD) simulations and found three of the compounds have stable interactions.

Resistance to existing drugs of tuberculosis bacteria demands an immediate requirement to develop effective new chemical entities acting on emerging targets. Seryl-tRNA synthetase (SerRS) is essential for the viability of Mycobacterium tuberculosis (MTB). In this study, we have attempted to develop the tertiary structure of SerRS through homology modelling and to elucidate the active site interactions of inhibitor compounds aided by docking. Homology modelling using PDB ID: '2DQ3: A' chain as template and validation of the model was carried out with Modeller V9.13 and SAVES online server respectively. About 1248 compounds from a putative kinase compound library of PubChem database found active in whole cell bioassay (AID2842) on MTB - H37Rv was used in docking studies using 'AutoDock'. Out of the tested molecules, nine showed docking scores ≤-10 kcal/mol with good drug-like properties were further subjected to molecular dynamics (MD) simulations and found three of the compounds have stable interactions.

Atomic-Level Protein Structure Refinement Using Fragment-Guided Molecular Dynamics Conformation Sampling

The quest for sustainable solutions to plastic pollution has driven research into plastic-degrading enzymes, offering promising avenues for polymer recycling applications. However, enzymes derived from natural sources often exhibit suboptimal thermostability, hindering their industrial viability. Protein engineering techniques have emerged as a powerful approach to enhance the desired properties of these biocatalysts. This study aims to conduct a comprehensive analysis of the thermostability of Vibrio palustris PETase (VpPETase) through an integrated computational approach encompassing homology modeling, site-specific molecular docking, molecular dynamics (MD) simulations, and comparative evaluation of a single-point mutation (V195F) against the wild-type enzyme. Homology modeling was used to predict VpPETase model using multiple templates. Model quality was rigorously assessed using Ramachandran plot analysis, ProSA, Verify 3D, and ERRAT. Molecular docking elucidated the catalytic region comprising residues His149, Asp117, and Ser71, while highlighting the pivotal roles of His149, Tyr1, and Ser71 in substrate binding affinity. MD simulations at various temperatures revealed higher stability at 313.15 K over a 100 ns trajectory, as evidenced by analyses of root-mean-square deviation (RMSD), radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen bonding, and root-mean-square fluctuation (RMSF). The V195F mutant exhibited a slight increase in stability compared to wild-type. While this study provides valuable insights into the thermostability of VpPETase, further investigations, including experimental validation of thermostability enhancements and in vitro characterization, are warranted to fully exploit the potential of this enzyme for industrial applications in plastic recycling.

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1 and 3beta-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His156 contributes to the 14-fold higher affinity that 3beta-HSD1 exhibits for substrate and inhibitor steroids compared with human 3beta-HSD2 containing Tyr156 in the otherwise identical catalytic domain. Our structural model of human 3beta-HSD localizes His156 or Tyr156 in the subunit interface of the enzyme homodimer. The model predicts that Gln105 on one enzyme subunit has a higher probability of interacting with His156 on the other subunit in 3beta-HSD1 than with Tyr156 in 3beta-HSD2. The Q105M mutant of 3beta-HSD1 (Q105M1) shifts the Michaelis-Menten constant (Km) for 3beta-HSD substrate and inhibition constants (Ki) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3beta-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3beta-HSD2. Our model also predicts that Gln240 in 3beta-HSD1 and Arg240 in 3beta-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate Km by 2.2-fold to a value similar to that of 3beta-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4-14-fold higher affinity profiles of 3beta-HSD1. Thus, key structural reasons for the substantially higher affinities of 3beta-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3beta-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.

Fusarium oxysporum causes significant economic losses in many crop plants by causing root rot, necrosis, and wilting symptoms. Homology and molecular dynamics studies are promising tools for the detection in F. oxysporum of the systemic resistance compound, salicylic acid, for control of the SKP1-CUL1-F-box protein complex. The structure of SKP1-CUL1-F-box subunit Skp1 from F. oxysporum is produced by Modeler 9v7 for the conduct of docking studies. The Skp1 structure is based on the yeast Cdc4/Skp1 (PDB ID: 3MKS A) crystal structure collected by the Protein data bank. Applying molecular dynamic model simulation methods to the final predicted structure and further evaluated by 3D and PROCHECK test programmers, the final model is verified to be accurate. Applying GOLD 3.0.1, SCF Complex Skp1 is used to prevent stress-tolerant operation. The SKP1-CUL1-F-box model is predicted to be stabilized and tested as a stable docking structure. The predicted model of the SCF structure has been stabilized and confirmed to be a reliable structure for docking studies. The results indicated that GLN8, LYS9, VAL10, TRP11, GLU48, ASN49 in SCF complex are important determinant residues in binding as they have strong hydrogen bonding with salicylic acid, which showed best docking results with SKP1-CUL1-F-box complex subunit Skp1 with docking score 25.25KJ/mol. Insilco studies have been used to determine the mode of action of salicylic acid for Fusarium control. Salicylic acid hinders the SKP1-CUL1-F-box complex, which is important in protein-like interactions through hydrogen bodings. Results from docking studies have shown that the best energy for SKP1-CUL1-F-box was salicylic acid. Communicated by Ramaswamy H. Sarma

The number of solved G-protein-coupled receptor (GPCR) crystal structures has expanded rapidly, but most GPCR structures remain unsolved. Therefore, computational techniques, such as homology modeling, have been widely used to produce the theoretical structures of various GPCRs for structure-based drug design (SBDD). Due to the low sequence similarity shared by the transmembrane domains of GPCRs, accurate prediction of GPCR structures by homology modeling is quite challenging. In this study, angiotensin II type I receptor (AT1R) was taken as a typical case to assess the reliability of class A GPCR homology models for SBDD. Four homology models of angiotensin II type I receptor (AT1R) at the inactive state were built based on the crystal structures of CXCR4 chemokine receptor, CCR5 chemokine receptor, and δ-opioid receptor, and refined through molecular dynamics (MD) simulations and induced-fit docking, to allow for backbone and side-chain flexibility. Then, the quality of the homology models was assessed relative to the crystal structures in terms of two criteria commonly used in SBDD: prediction accuracy of ligand-binding poses and screening power of docking-based virtual screening. It was found that the crystal structures outperformed the homology models prior to any refinement in both assessments. MD simulations could generally improve the docking results for both the crystal structures and homology models. Moreover, the optimized homology model refined by MD simulations and induced-fit docking even shows a similar performance of the docking assessment to the crystal structures. Our results indicate that it is possible to establish a reliable class A GPCR homology model for SBDD through the refinement by integrating multiple molecular modeling techniques.

Protein thermostability can be increased by some glycine to proline mutations in a target protein. However, not all glycine to proline mutations can improve protein thermostability, and this method is suitable only at carefully selected mutation sites that can accommodate structural stabilization. In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved by the experiments. The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 (Ochr-MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure. A profile of the root mean square fluctuations of Ochr-MPH was calculated at the nanosecond timescale, and an eight-amino acid loop region (residues 186-193) was identified as having high conformational fluctuation. The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity. The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P/G198P) were modeled and analyzed using molecular dynamics simulations. The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr-MPH. The thermostability and kinetic behavior of the wild-type and three mutant enzymes were measured. The results were consistent with the computational predictions, and the mutant G194P was found to have higher thermostability than the wild-type enzyme.

Proline utilization A (PutA) is a large, bifunctional enzyme composed of FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent L-glutamate γ-semialdehyde dehydrogenase (GSALDH) domains. The sequential actions of PRODH and GSALDH catalyze proline catabolism, i.e., the 4-electron oxidation of L-proline to L-glutamate via the intermediates Δ1-pyrroline-5-carboxylate (P5C) and L-glutamate γ-semialdehyde (GSAL). Crystal structures of PutAs have revealed a conserved, 42 Å long tunnel connecting the two active sites, implying a substrate channeling mechanism, which has been confirmed with kinetic measurements. PutAs serve as excellent model systems for studying the structural basis, dynamics, and mechanism of substrate channeling. Here we used kinetic and conventional crystallography to trap transient intermediates and conformational states along the PutA catalytic cycle. For kinetic crystallography, crystals of Sinorhizobium meliloti PutA (SmPutA) were soaked in the substrates of the PRODH reaction to generate P5C and GSAL. Kinetic crystallography of SmPutA site-directed mutant variants designed to slow specific steps of the reaction generated the first snapshots of a heretofore unknown transient intermediate of the PRODH reaction consisting of P5C covalently connected to the N5 atom of FAD (1.9 Å, Rwork/Rfree = 0.17/0.21), the aldehyde substrate GSAL bound in the GSALDH site (1.5 Å, Rwork/Rfree = 0.18/0.21), and the covalent acyl-enzyme intermediate of the GSALDH reaction (1.7 Å, Rwork/Rfree = 0.22/0.26). Conventional crystallography was also used to capture the structure of SmPutA complexed with the final product of proline catabolism, L-glutamate (1.5 Å, Rwork/Rfree = 0.17/0.19). Complementary all-atom, μs-scale molecular dynamics (MD) simulations provide insight into molecular motions - both protein and water - associated with substrate channeling. Water diffusion in the substrate channeling tunnel appears to be much slower than bulk solvent, and tracking of waters during MD reveals vulnerable parts of the tunnel that may allow the intermediates P5C/GSAL to escape to the bulk solvent, providing an explanation for the “leaky channeling” observed in kinetic experiments. Co-opting “static” protein crystals to perform catalysis through kinetic crystallography, combined with computational descriptions of the dynamics of the captured catalytic states, offers multi-faceted insight into how substrate channeling is achieved in PutAs.

Two-pore domain potassium (K2P) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K2P channels. We describe A1899 as a potent and highly selective blocker of the K2P channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K2P open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K2P channels.

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 A above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp(98). Furthermore, the 7-position of the imipramine ring is found vicinal to Phe(335), and the pocket lined by Ala(173) and Thr(439) is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).