Investigation of the Effects of Mutating Iron-Coordinating Residues in Rieske Dioxygenases

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 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.

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.

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.

Asymmetric hydrogenations are important reactions in industrial synthesis, as up to two stereogenic centres can be generated. Given the current trend towards more “green” or sustainable chemistry, biocatalytic approaches are becoming more important in the production of fine chemicals, pharmaceuticals, and agrochemical products. There are at least four known classes of enzymes that have been explored for their biocatalytic applicability, collectively known as ene-reductases (ERs). Each enzyme requires NAD(P)H as the hydride donor, whereas the mechanism, substrate scope and product stereoand/or enantiospecificity differs between biocatalyst classes. The most widely investigated class is the old yellow enzyme (OYE; EC 1.6.99.1) family and many recent reviews have summarised the (potential) applications of these enzymes for industrial syntheses, for example, Refs. [1–3] . These flavin mononucleotide (FMN)-containing enzymes catalyse the C=C reduction of a wide variety of a,b-unsaturated aldehydes, ketones, nitroalkenes, maleimides, dicarboxylic acids and their esters, and nitriles. A second less well-characterised enzyme class catalysing biocatalytic reductions are enoate reductases (EC 1.3.1.31). These oxygen-sensitive enzymes contain both FAD and [4Fe-4S], and are members of the NADH:flavin oxidoreductase/NADH oxidase family. They have been shown to catalyse the reduction of a variety of a,b-unsaturated monoacids and esters. 5] A more recent class of enzymes investigated are the medium-chain dehydrogenase/reductase (MDR) family of oxidoreductases (1.3.1.-). For example, the flavin-independent double-bond reductase from Nicotiana tabacum (NtDBR) catalyses the reduction of a wide variety of a,b-unsaturated aldehydes, ketones and nitroalkenes. There are a few examples in the literature of potential applications of a fourth class of enzymes, namely the flavin-independent short-chain dehydrogenase/reductases (SDR) from plants. Examples include two menthol dehydrogenases, ( )-menthol dehydrogenase (EC 1.1.1.207) and (+)-neomenthol dehydrogenase (EC 1.1.1.208), which catalyse the reduction of specific menthone isomers to menthol. Unfortunately, the high cost of NAD(P)H nicotinamide coenzymes makes them uneconomical for industrial-scale syntheses. Therefore, alternative methodologies have been adopted to supply the necessary hydride equivalents for alkene reduction. One such method is to include a coenzyme recycling system, in which catalytic levels of NAD(P) are constantly regenerated to NAD(P)H. Several systems have been developed and routinely employed for ER biocatalytic reactions, such as glucose dehydrogenase, glucose-6-phosphate dehydrogenase (G6PDH/glucose-6-phosphate), formate dehydrogenase and phosphite dehydrogenase, and reviewed elsewhere. In each case, only catalytic levels of NAD(P) are needed but stoichiometric levels of a co-substrate are required to drive the recycling enzyme. Faber and coworkers have performed comparative biotransformations by using multiple cofactor recycling systems, substrates and ERs, to determine the ideal hydride source. Product yield and/or enantioselectivity can vary considerably, depending on which coenzyme recycling system has been employed. Additionally, the inclusion of a second enzyme system into large-scale (bio)synthesis may present problems, for example in maintenance of the activity and stability of two enzymes and the high cost of some co-substrates. Electrochemical regeneration of nicotinamide coenzymes has also been investigated, for example in the direct cathodic reduction of NAD(P) . Unfortunately, this simple regeneration method is hampered by low selectivity and the formation of undesired NAD(P) dimeric side products. Several techniques have been developed to bypass the need for nicotinamide coenzymes by using alternative hydride donors to reduce the flavin cofactor of some ERs. Reetz and coworkers described a method whereby the FMN cofactor of the OYE homologue YqjM from Bacillus subtilis was photoreduced, employing free flavin and a sacrificial electron donor. The YqjM bound oxidised FMN was reduced by the free photoreduced FMN, thereby generating the active enzyme. This NAD(P)H-free system was successful in converting ketoisophorone to (R)-levodione, albeit with lower product enantiopurity. Similarly, Hollmann and co-workers described the photoenzymatic flavin reduction of YqjM and NEMR (N-ethylmalemide reductase from E. coli) by using alternative sacrificial electron donors formate and phosphite. These methods required strict anaerobic conditions to prevent the rapid reoxidation of reduced FMN by molecular oxygen. Early attempts at NAD(P)H-independent alkene reduction focussed on the use of artificial mediators, such as N,N-dimethyl4-4-bipyridinium [methyl viologen (MV)] , as a means of reducing the flavin in clostridial enoate reductases. A variety of reduced mediator recycling systems were employed, including direct electron transfer from a cathode and enzymatic-based recycling systems. Successful enzyme-coupled mediator recycling was achieved used the following systems: 1) hydroge[a] Dr. H. S. Toogood, Dr. T. Knaus, Prof. N. S. Scrutton Manchester Institute of Biotechnology, Faculty of Life Sciences University of Manchester 131 Princess Street, Manchester M1 7DN (UK) Fax: (+44)01613068918 E-mail : nigel.scrutton@manchester.ac.uk

An enhanced toluene dioxygenase platform for the production of cis-1,2-dihydrocatechol in Escherichia coli BW25113 lacking glycerol dehydrogenase activity

The Rieske dioxygenases are a group of non-heme iron enzymes, which catalyze the stereospecific cis-dihydroxylation of its substrates. Herein, we report the iron(II) coordination chemistry of the ligands 3,3-bis(1-methylimidazol-2-yl)propionate (L1) and its neutral propyl ester analogue propyl 3,3-bis(1-methylimidazol-2-yl)propionate (PrL1). The molecular structures of two iron(II) complexes with PrL1 were determined and two different coordination modes of the ligand were observed. In [Fe(II)(PrL1)(2)](BPh(4))(2) (3) the ligand is facially coordinated to the metal with an N,N,O donor set, whereas in [Fe(II)(PrL1)(2)(MeOH)(2)](OTf)(2) (4) a bidentate N,N binding mode is found. In 4, the solvent molecules are in a cis arrangement with respect to each other. Complex 4 is a close structural mimic of the crystallographically characterized non-heme iron(II) enzyme apocarotenoid-15-15'-oxygenase (APO). The mechanistic features of APO are thought to be similar to those of the Rieske oxygenases, the original inspiration for this work. The non-heme iron complexes [Fe(II)(PrL1)(2)](OTf)(2) (2) and [Fe(II)(PrL1)(2)](BPh(4))(2) (3) were tested in olefin oxidation reactions with H(2)O(2) as the terminal oxidant. Whereas 2 was an active catalyst and both epoxide and cis-dihydroxylation products were observed, 3 showed negligible activity under the same conditions, illustrating the importance of the anion in the reaction.

Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). The enzyme consisted of three protein components: NADH-ferredoxintol oxidoreductase (reductasetol), ferredoxintol, and a terminal oxygenase which is an iron-sulfur protein (ISPtol). Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT). Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction. Colonies of reductasetol mutants were white or white with a light blue center, ferredoxintol strains were light blue with a dark blue center, and strains that lacked ISPtol gave dark blue colonies. Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction. Strains lacking all three components appeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component. Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.

Rational and mechanistic approaches for improving biocatalyst performance

Cytochrome P450 monooxygenases (P450s) are some of nature's most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C-H bonds has led to their increasing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding experiments, steady-state kinetics studies, and X-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity.

Cells of Pseudomonas putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methyl-cyclohexa-3,5-diene. One of these components has been purified to homogeneity and shown to be a flavoprotein that contains FAD as the only detectable prosthetic group. Fad was removed from the enzyme during purification. However, equilibrium dialysis experiments showed that the enzyme can bind one mol of FAD/mol of enzyme protein. The apparent molecular weight of the enzyme is 46,000, as judged by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. The latter result suggests the presence of a single polypeptide chain. The amino acid composition of the enzyme reveals a relatively high content of the hydrophobic amino acids leucine, isoleucine, and valine and is remarkably similar in composition to the flavoproteins that function in certain monooxygenase enzyme systems. The purified enzyme catalyzes the reduction of dichloroindophenol, nitrobluetetrazolium, ferricyanide, and ferredoxinTOL. Its ability to reduce cytochrome c and to function in the toluene dioxygenase enzyme system is absolutely dependent on the presence of ferredoxinTOL.

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The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174-5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429).

Members of the cytochrome P450 superfamily of monooxygenases (P450s) are some of nature's most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize C–H bonds has led to their growing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present work, we biochemically and structurally characterized diverse bacterial P450s involved in the biosynthesis of 16-membered ring macrolide antibiotics with significant potential for development into robust biocatalysts for the late-stage functionalization of complex molecules. Following exploratory efforts to probe the reactivity properties of the P450 MycCI from the mycinamicin biosynthetic pathway, we discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding and kinetics experiments along with X-ray crystallographic analysis of MycCI bound to its native substrate. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared with that of MycCI. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI exhibits a strict preference for 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. These results were partially explained through analysis of the X-ray crystal structure of TylHI in complex with its native substrate together with biochemical characterization of several sitedirected mutants. Comparative analysis of the MycCI/TylHI homolog ChmHI from the chalcomycin biosynthetic pathway provided a basis for constructing MycCI/TylHI chimeras in order to gain further insight into the features dictating the differences in the reactivity profiles of these two related P450s. These experiments unveiled the central role of the BC loop region in influencing the binding of 16-membered ring substrates to MycCI and TylHI. Overall, our studies have shed light on the molecular-level details underpinning the unique catalytic divergence of two homologous biosynthetic P450s and point toward specific regions in each protein that may be targeted in future efforts to rationally engineer novel P450-based biocatalysts. Support or Funding Information NSF under the CCI Center for Selective C–H Functionalization (CHE-1205646 and CHE-1700982), NIH (R01-GM078553, R35-GM118101, and T32-GM008353), University of Michigan Rackham Predoctoral Fellowship Physiological reactions catalyzed by P450s MycCI and TylHI in their respective biosynthetic pathways. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.