Front Cover Picture: Semi‐Rational Engineering of Toluene Dioxygenase from Pseudomonas putida F1 towards Oxyfunctionalization of Bicyclic Aromatics (Adv. Synth. Catal. 21/2021)

Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent. With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.

Synthesis, characterisation and catalytic performance of boron substituted SBA-15 molecular sieves

Photochemical oxidation of hydrocarbons with molecular oxygen is potentially an environmentally benign method for the selective oxidation of hydrocarbons. In this study, in situ FT-IR spectroscopy and ex situ NMR spectroscopy were used to investigate the factors that influence product formation and selectivity in the room-temperature photooxidation of 1-alkenes in zeolites. Upon irradiation with broadband visible light, propylene, 1-butene, and 1-pentene loaded in BaY were photooxidized with molecular oxygen. As discussed in the literature, initial excitation of alkene and molecular complexes results in the selective formation of unsaturated aldehydes and ketones, proposed to occur through a hydroperoxide intermediate. In addition, epoxide and alcohol products are formed when the hydroperoxide intermediate reacts with an unreacted parent alkene molecule. Here it is shown that saturated aldehydes and ketones are formed as well through both a thermal ring-opening reaction of the epoxide in BaY and a second photochemical oxidation route involving a dioxetane intermediate. The yield of saturated aldehydes and ketones increased with decreasing wavelength, increasing temperature, and at a given temperature and wavelength, increasing chain length. Photooxidation of propylene in BaX, BaZSM-5, and BaBeta zeolites was also investigated. Photooxidation in BaX is very similar to that of BaY. In zeolites, BaZSM-5 and BaBeta, propylene polymerized upon adsorption. The polymer, polypropylene, also undergoes photooxidation with molecular oxygen to form an oxygenated polymer product. The results of this study show that product formation and selectivity in the photooxidation of 1-alkenes in zeolites depends on several factors. These factors include thermal reactions of the reactant and photoproduct molecules in the zeolite at ambient temperatures. Several reactions of 1-alkenes in cation-exchanged zeolites contribute to the loss of selectivity; they include: epoxide ring opening, double-bond migration, and alkene polymerization. Some of these reactions are proposed to occur at Brønsted acid sites that are present in various amounts in cation-exchanged zeolites.

The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.

Toluene dioxygenase (TDO) from Pseudomonas putida F1 was engineered towards the oxyfunctionalization of bicyclic substrates. Single and double mutant libraries addressing 27 different positions, located at the active site and entrance channel were generated. In total, 176 different variants were tested employing the substrates naphthalene, 1,2,3,4‐tetrahydroquinoline, and 2‐phenylpyridine. Introduced mutations in positions M220, A223 and F366, exhibited major influences in terms of product formation, chemo‐, regio‐ and enantioselectivity. By semi‐rational evolution, we lighted up the TDO capability to convert bulkier substrates than its natural substrate, at unprecedented reported conversions. Thus, the most active TDO variants were applied to biocatalytic oxyfunctionalizations of 1,2,3,4‐tetrahydroquinoline, and 2‐phenylpyridine, enabling the production of substantial amounts of (+)‐(R)‐1,2,3,4‐tetrahydroquinoline‐4‐ol (71% isolated yield, 94% ee) and (+)‐(1S,2R)‐3‐(pyridin‐2‐yl)cyclohexa‐3,5‐diene‐1,2‐diol (60% isolated yield, 98% ee), respectively. Here, we provide a set of novel TDO‐based biocatalysts useful for the preparation of oxyfunctionalized bicyclic scaffolds, which are valuable to perform downstream synthetic processes.magnified image

BackgroundThe benzoylformate decarboxylase (BFD) from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR) as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS).ResultsAlthough the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro) had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala) showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln) behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences in metabolic activity of their respective host strain.ConclusionsThe BFD-variants with high cofactor binding affinity (wild type, His281Ala, Ser181Thr) obviously extract thiamine from the medium and bind it tightly to the enzyme. This might explain the hampered growth of these clones. In contrast, growth of clones expressing variants with low cofactor binding affinity (Leu476His, Leu476Pro, Leu476Pro-Ser181Thr) is not impaired. Leu476Gln has an intermediate cofactor binding strength, thus, growth of its host strain depends on the specific cultivation conditions. This paper shows that slight differences of the amino acid composition can affect protein expression and cultivation and might require an adaptation of media components. Effects such as the observed are hardly foreseeable and difficult to detect in conventional screening processes. Via small scale experiments with on-line measurements in shake flasks such effects influencing the cultivation and product formation can be detected and avoided.

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high-cell-density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth-limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ∼30 and ∼90 mg L(-1), respectively. Remarkably, a little increase of mcl-PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high-cell-density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl-PHA content occurred up to a DCT of ∼540 mg L(-1). However, specific growth rate exhibited a significant decrease, indicating that successful high-cell-density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed-batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl-PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl-PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L(-1)h(-1) (at 3.2 bar).

An important challenge in electrochemical CO2 reduction (ECR) is relating experimental conditions to their consequences, particularly in terms of product selectivity. The problem lies in the lack of descriptors which adequately describe the experimental protocols and their associated results. In this study, a machine learning approach is applied to correlate the molar composition of 21 single metals and 23 bimetallic particles, as well as operating parameters, from a large collection of synthetic records compiled from the literature with product selectivity. The decision tree obtained shows the conditions that lead to high desired product selectivity and provides a heuristic insight into its electrochemistry. As such, the data does not provide details. However, machine learning algorithms are capable of identifying hidden patterns in the data, providing a deeper insight into the chemistry involved in product formation in the ECR.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRelations among enzymes of the β-ketoadipate pathway. III. Properties of crystalline γ-carboxymuconolactone decarboxylase from Pseudomonas putidaDonna Parke, Richard B. Meagher, and L. Nicholas OrnstonCite this: Biochemistry 1973, 12, 18, 3537–3542Publication Date (Print):August 1, 1973Publication History Published online1 May 2002Published inissue 1 August 1973https://doi.org/10.1021/bi00742a029RIGHTS & PERMISSIONSArticle Views77Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (3 MB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTInitial reactions in the oxidation of ethylbenzene by Pseudomonas putidaD. T. Gibson, Brigitte Gschwendt, W. K. Yeh, and V. M. KobalCite this: Biochemistry 1973, 12, 8, 1520–1528Publication Date (Print):April 1, 1973Publication History Published online1 May 2002Published inissue 1 April 1973https://doi.org/10.1021/bi00732a008RIGHTS & PERMISSIONSArticle Views302Altmetric-Citations103LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (927 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMandelate racemase from Pseudomonas putida. Subunit composition and absolute divalent metal ion requirementJudy A. Fee, George D. Hegeman, and George L. KenyonCite this: Biochemistry 1974, 13, 12, 2528–2532Publication Date (Print):June 1, 1974Publication History Published online1 May 2002Published inissue 1 June 1974https://doi.org/10.1021/bi00709a008RIGHTS & PERMISSIONSArticle Views130Altmetric-Citations37LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to Degradation, Phytoprotection and Phytoremediation of Phenanthrene by Endophyte Pseudomonas putida, PD1Zareen Khan, David Roman, Trent Kintz, May delas Alas, Raymond Yap, and Sharon Doty*Cite this: Environ. Sci. Technol. 2014, 48, 22, 13557Publication Date (Web):November 7, 2014Publication History Published online7 November 2014Published inissue 18 November 2014https://doi.org/10.1021/es505252vCopyright © 2014 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views487Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (112 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOxidative degradation of aromatic hydrocarbons by microorganisms. III. Formation of (+)-cis-2,3-dihydroxy-1-methyl-4,6-cyclohexadiene from toluene by Pseudomonas putidaDavid T. Gibson, M. Hensley, H. Yoshioka, and T. J. MabryCite this: Biochemistry 1970, 9, 7, 1626–1630Publication Date (Print):March 31, 1970Publication History Published online1 May 2002Published inissue 31 March 1970https://doi.org/10.1021/bi00809a023RIGHTS & PERMISSIONSArticle Views492Altmetric-Citations347LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (506 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAbsolute stereochemistry of the (+)-cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene produced from toluene by Pseudomonas putidaHerman. Ziffer, Donald M. Jerina, David T. Gibson, and Val M. KobalCite this: J. Am. Chem. Soc. 1973, 95, 12, 4048–4049Publication Date (Print):June 1, 1973Publication History Published online1 May 2002Published inissue 1 June 1973https://doi.org/10.1021/ja00793a036RIGHTS & PERMISSIONSArticle Views137Altmetric-Citations69LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (288 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNew oxygenases in the degradation of flavones and flavanones by Pseudomonas putidaEmeric Schultz, F. E. Engle, and J. M. WoodCite this: Biochemistry 1974, 13, 8, 1768–1776Publication Date (Print):April 1, 1974Publication History Published online1 May 2002Published inissue 1 April 1974https://doi.org/10.1021/bi00705a033RIGHTS & PERMISSIONSArticle Views162Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (862 KB) Get e-Alerts Get e-Alerts