Improvement of Rieske Dioxygenase Activity Through Remodeling of the Substrate Tunnel

Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high-value compounds, due to their capacity to perform the cis-dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for the cis-dihydroxylation of benzoates. Through rational mutagenesis and screening, TDO variants with substantially improved activity over the wild-type enzyme were identified. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in an effort to elucidate how the identified mutations resulted in improved activity for this polar substrate class. These analyses revealed modification of the substrate tunnel as the likely cause of 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.

Simulated Mechanism of Triclosan in Modulating the Active Site and Loop of FabI by Computer

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.

Herein we report the development of a new periodate-based reactive assay system for the fluorescent detection of the cis-diol metabolites produced by Rieske dioxygenases. This sensitive and diastereoselective assay system successfully evaluates the substrate scope of Rieske dioxygenases and determines the relative activity of a rationally designed Rieske dioxygenase variant library. The high throughput capacity of the assay system enables rapid and efficient substrate scope investigations and screening of large dioxygenase variant libraries.

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl’s side chain tightens and relaxes the opening to the channel in conjunction with the arginyl’s, though the latter’s side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.

Squalene epoxidase plays a pivotal role in the biosynthesis of ergosterol, its derivatives, and other triterpenoid compounds by catalyzing the transformation of squalene into 2,3-oxidosqualene. However, its low catalytic efficiency remains a primary bottleneck for the microbial synthesis of triterpenoids. In this study, the catalytic activity of the squalene epoxidase from Saccharomyces cerevisiae was significantly improved by reshaping its substrate tunnel, resulting in a marked increase in the yield of the final product, ergosterol. First, the amino acid in the catalytic pocket of squalene epoxidase was replaced with alanine (Ala), effectively reducing the steric hindrance, and thus, enhancing the affinity of the enzyme with its substrate. Then, the V249H/L343A mutant was obtained by redesigning the substrate tunnel of dominant mutant L343A, thus, increasing the titer of ergosterol. The study also elucidated the mechanism behind the increased catalytic activity of the V249H/L343A mutant through substrate tunnel parameter analysis and molecular dynamics simulations. Finally, a titer of 3345 mg/L of ergosterol was achieved by strains containing V249H/L343A in a 5 L bioreactor, with a specific yield of 84 mg/g dry cell weight (DCW), marking a 64% increase compared with the titer achieved by wild type strains. This study established a strong foundation for improving the synthetic efficiency of ergosterol and other triterpenoid compounds.

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

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.

Molecular selection, ion exclusion, and water permeation are well known regulatory mechanisms in aquaporin. Water permeability was found to be diverse in different subgroups of plasma membrane intrinsic proteins (PIPs), even though the residues surrounding the water holes remained the same across the subgroups. Upon homology modeling and structural comparison, a conserved Ala/Ile(Val) residue difference was identified in helix 2 that affected the conformation of the NPA region and consequently influenced the water permeability. The residue difference was found to be conservative within the two subgroups of PIPs in rice as well as in other plants. Functional tests further confirmed the prediction via site-directed mutagenesis where replacement of Ala(103) or Ala(102) in respective OsPIP1;1 or OsPIP1;3 with Val yielded 7.0- and 2.2-fold increases in water transportation, and substitution of Ile(98) or Val(95) in respective OsPIP2;3 or OsPIP2;7 with Ala resulted in 73 or 52% reduction of water transportation. Based on structural analyses and molecular dynamics simulations, we proposed that the difference in water permeability was attributed to the orientation variations of helix 2 that modified water-water and water-protein interactions.

Mechanisms of persimmon pectin methyl esterase activation by high pressure processing based on chemical experiments and molecular dynamics simulations