Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase

Biological carbon dioxide (CO₂) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO₂ at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO₂ and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.     

Fabrication of a Thermoresponsive Biohybrid Double Hydrophilic Block Copolymer by a Cofactor Reconstitution Approach

The fabrication of a thermoresponsive biohybrid double hydrophilic block copolymer (DHBC) by a cofactor reconstitution approach is reported. Poly(N‐isopropylacrylamide) (PNIPAM) bearing a porphyrin moiety at the chain terminal, PPIXZn‐PNIPAM, is synthesized by the combination of ATRP and a click reaction. The subsequent cofactor reconstitution process between apomyoglobin and PPIXZn‐PNIPAM affords well‐defined myoglobin‐b‐PNIPAM protein–polymer bioconjugates. Behaving as typical responsive DHBCs, the obtained myoglobin‐b‐PNIPAM biohybrid diblock copolymer exhibits thermo‐induced aggregation behavior in aqueous solution as a result of the presence of the thermoresponsive PNIPAM block, as revealed by temperature‐dependent transmittance, dynamic laser light scattering measurements, transmission electron microscopy, and scanning electron microscopy. This work represents the first report of the preparation of responsive biohybrid DHBCs by the cofactor reconstitution process.

     

Synthesis of a pyrrolo annulated pyrido[2,3-D]pyrimidine as a potential nonclassical antifolate

Cyclocondensation of 2,4,6-triaminopyrimidine ( 4 ) with ethyl N-benzyl-4-oxo-3-pyrrolidine carboxylate ( 5 ) in diphenyl ether regiospecifically afforded a new tricyclic, angular 1,3,8-trisubstituted pyrrolo[3′,4′:4,5]-pyrido[2,3-d]pyrimidine-6-one 1 in excellent yield. The ketoester 5 was prepared by a literature method. Compound 1 in addition to being a new heterocyclic system is an important key precursor to a variety of classical and nonclassical tricyclic, 5-deaza analogues of the folate cofactor 5,10-methylenetetrahydrofolate 3 .     

Regioselective para‐Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase

The utilization of CO₂ as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme‐catalyzed para ‐carboxylation of catechols, employing 3,4‐dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN^iminium species. This study reports on the in vitro reconstitution and activation of a prFMN‐dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN‐associated 1,3‐dipolar cycloadditions in related enzymes.     

Identification of HcgC as a SAM‐Dependent Pyridinol Methyltransferase in [Fe]‐Hydrogenase Cofactor Biosynthesis

Previous retrosynthetic and isotope‐labeling studies have indicated that biosynthesis of the iron guanylylpyridinol (FeGP) cofactor of [Fe]‐hydrogenase requires a methyltransferase. This hypothetical enzyme covalently attaches the methyl group at the 3‐position of the pyridinol ring. We describe the identification of HcgC, a gene product of the hcgA‐G cluster responsible for FeGP cofactor biosynthesis. It acts as an S‐adenosylmethionine (SAM)‐dependent methyltransferase, based on the crystal structures of HcgC and the HcgC/SAM and HcgC/S‐adenosylhomocysteine (SAH) complexes. The pyridinol substrate, 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol, was predicted based on properties of the conserved binding pocket and substrate docking simulations. For verification, the assumed substrate was synthesized and used in a kinetic assay. Mass spectrometry and NMR analysis revealed 6‐carboxymethyl‐3,5‐dimethyl‐4‐hydroxy‐2‐pyridinol as the reaction product, which confirmed the function of HcgC.     

Precise Design of Artificial Cofactors for Enhancing Peroxidase Activity of Myoglobin: Myoglobin Mutant H64D Reconstituted with a “Single‐Winged Cofactor” Is Equivalent to Native Horseradish Peroxidase in Oxidation Activity

H64D myoglobin mutant was reconstituted with two different types of synthetic hemes that have aromatic rings and a carboxylate‐based cluster attached to the terminus of one or both of the heme‐propionate moieties, thereby forming a “single‐winged cofactor” and “double‐winged cofactor,” respectively. The reconstituted mutant myoglobins have smaller K_m values with respect to 2‐methoxyphenol oxidation activity relative to the parent mutant with native heme. This suggests that the attached moiety functions as a substrate‐binding domain. However, the k_cat value of the mutant myoglobin with the double‐winged cofactor is much lower than that of the mutant with the native heme. In contrast, the mutant reconstituted with the single‐winged cofactor has a larger k_cat value, thereby resulting in overall catalytic activity that is essentially equivalent to that of the native horseradish peroxidase. Enhanced peroxygenase activity was also observed for the mutant myoglobin with the single‐winged cofactor, thus indicating that introduction of an artificial substrate‐binding domain at only one of the heme propionates in the H64D mutant is the optimal engineering strategy for improving the peroxidase activity of myoglobin.     

The nature of active centers of a polymeric catalase analog

The decomposition of hydrogen peroxide by a polymer complex of Fe³⁺ and poly(acrylic acid) partially amidated by diethylenetriamine has been studied. The molecular mechanism of catalysis is discussed. The rate constant of the reaction is k = 6.9 × 10⁷ exp {?2.300/RT}. Decomposition of hydrogen peroxide proceeds via activation by the diethylenetriamine which is a cofactor.     

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

A tandem enzymatic strategy to enhance the scope of C‐alkylation of small molecules via the in situ formation of S‐adenosyl methionine (SAM) cofactor analogues is described. A solvent‐exposed channel present in the SAM‐forming enzyme SalL tolerates 5′‐chloro‐5′‐deoxyadenosine (ClDA) analogues modified at the 2‐position of the adenine nucleobase. Coupling SalL‐catalyzed cofactor production with C‐(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C‐(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants that influence C‐alkylation provides the basis to develop a late‐stage enzymatic platform for the preparation of high value small molecules.     

5,10-Methylene-5,6,7,8-tetrahydrofolate conformational transitions upon binding to thymidylate synthase: molecular mechanics and continuum solvent studies

Summary We applied the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) approach to evaluate relative stability of the extended (flat) and C-shaped (bent) solution conformational forms of the 5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) molecule in aqueous solution. Calculations indicated that both forms have similar free energies in aqueous solution but detailed energy components are different. The bent solution form has lower intramolecular electrostatic and van der Waals interaction energies. The flat form has more favorable solvation free energy and lower contribution from the bond, angle and torsion angle molecular mechanical internal energies. We exploit these results and combine them with known crystallographic data to provide a model for the progressive binding of the mTHF molecule, a natural cofactor of thymidylate synthase (TS), to the complex forming in the TS-catalyzed reaction. We propose that at the time of initial weak binding in the open enzyme the cofactor molecule remains in a close balance between the flat and bent solution conformations, with neither form clearly favored. Later, thymidylate synthase undergoes conformational change leading to the closure of the active site and the mTHF molecule is withdrawn from the solvent. That effect shifts the thermodynamic equilibrium of the mTHF molecule toward the bent solution form. At the same time, burying the cofactor molecule in the closed active site produces numerous contacts between mTHF and protein that render change in the shape of the mTHF molecule. As a result, the bent solution conformer is converted to more strained L-shaped bent enzyme conformer of the mTHF molecule. The strain in the bent enzyme conformation allows for the tight binding of the cofactor molecule to the productive ternary complex that forms in the closed active site, and facilitates the protonation of the imidazolidine N10 atom, which promotes further reaction. *To whom correspondence should be addressed. Fax: +4822-822-5342; E-mail: a.jarmula@nencki.gov.pl     

Flavin Redox Bifurcation as a Mechanism for Controlling the Direction of Electron Flow during Extracellular Electron Transfer

The iron‐reducing bacterium Shewanella oneidensis MR‐1 has a dual directional electronic conduit involving 40 heme redox centers in flavin‐binding outer‐membrane c‐type cytochromes (OM c‐Cyts). While the mechanism for electron export from the OM c‐Cyts to an anode is well understood, how the redox centers in OM c‐Cyts take electrons from a cathode has not been elucidated at the molecular level. Electrochemical analysis of live cells during switching from anodic to cathodic conditions showed that altering the direction of electron flow does not require gene expression or protein synthesis, but simply redox potential shift about 300 mV for a flavin cofactor interacting with the OM c‐Cyts. That is, the redox bifurcation of the riboflavin cofactor in OM c‐Cyts switches the direction of electron conduction in the biological conduit at the cell–electrode interface to drive bacterial metabolism as either anode or cathode catalysts.     

The Role of Pyridoxal Phosphate in the Catalysis of Glycogen Phosphorylases

Glycogen phosphorylases catalyze the degradation of glycogen by phosphate (or arsenate) to glucose 1-phosphate (or glucose + arsenate). All glycogen phosphorylases that have been studied so far contain pyridoxal 5′-phosphate, a vitamin B₆-derivative, as cofactor. Removal of the cofactor results in an inactive apoenzyme. However, reduction of the azomethine bond linking pyridoxal phosphate to an ?-aminolysyl side chain of the enzyme with NaBH₄ does not inactivate glycogen phosphorylase. If therefore the cofactor should be involved in catalysis in glycogen phosphorylase it must function differently from all other classical pyridoxal phosphate dependent enzymes, for these are inactivated by reduction. ³¹P-NMR spectroscopy has revealed that the 5′-phosphate group of pyridoxal phosphate is present in catalytically active forms of glycogen phosphorylases as dianion in a hydrophobic environment shielded from aqueous solvent. Covalent and/or allosteric activation of muscle glycogen phosphorylases is accompanied by a transition of the monoprotonated form to the dianionic form of the phosphate group of the cofactor. We now report on such ionization changes in unregulated active potato- and E. coli maltodextrin phosphorylases on binding of glucose and oligosaccharides and following catalytic turnover, i.e. arsenolysis of α-1,4-glycosidic bonds. (Like glycogen phosphorylases, maltodextrin phosphorylases belong to the class of α-glucan phosphorylases.) The results of experiments carried out by our group together with recent findings on the three dimensional structure of crystalline muscle glycogen phosphorylases indicate a participation of the dianionic phosphate group as proton acceptor for the glucosyl transfer to and from the glucosyl acceptor. Although other interpretations are not excluded, at present little doubt remains that in the case of glycogen phosphorylases the dianionic phosphate group of the cofactor functions in catalysis.     

Unique peculiarities of the structure of the Fe-Mo cofactor of nitrogenase favorable for multicenter coordination of a nitrogen molecule

The probable coordination mode of a nitrogen molecule on the Fe-Mo cofactor of nitrogenase has been theoretically considered, taking into account both the well-known data on the structure of the Fe-Mo cofactor and the substrate selectivity of nitrogenase and the results of semiempirical calculations of the electronic structures of the cofactor and its complexes with molecular nitrogen. The distances between the Fe atoms in the cofactor are favorable for different multicenter coordination modes of a nitrogen molecule: above the Fe₄ face along its diagonal, through this face, and inside the Fe₆ prism perpendicularly to its axes. It is important that the nitrogen atoms are open for protonation in all coordination modes. The first mode is disadvantageous due to steric hindrances. Of the other variants, the latter is the most favorable both energetically and from the viewpoint of weakening of the N-N bond.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1928–1933, August, 1996.     

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli. Biochemical and spectroscopic analyses demonstrate the integrity of the heterologously expressed NifEN in composition and functionality and, additionally, the ability of an engineered NifEN variant to mimic NifDK in retaining the matured cofactor at an analogous cofactor‐binding site. This is an important step toward piecing together a viable pathway for the heterologous expression of nitrogenase and identifying variants for the mechanistic investigation of this enzyme.     

Exploiting Cofactor Versatility to Convert a FAD‐Dependent Baeyer–Villiger Monooxygenase into a Ketoreductase

Cyclohexanone monooxygenases (CHMOs) show very high catalytic specificity for natural Baeyer–Villiger (BV) reactions and promiscuous reduction reactions have not been reported to date. Wild‐type CHMO from Acinetobacter sp. NCIMB 9871 was found to possess an innate, promiscuous ability to reduce an aromatic α‐keto ester, but with poor yield and stereoselectivity. Structure‐guided, site‐directed mutagenesis drastically improved the catalytic carbonyl‐reduction activity (yield up to 99 %) and stereoselectivity (ee up to 99 %), thereby converting this CHMO into a ketoreductase, which can reduce a range of differently substituted aromatic α‐keto esters. The improved, promiscuous reduction activity of the mutant enzyme in comparison to the wild‐type enzyme results from a decrease in the distance between the carbonyl moiety of the substrate and the hydrogen atom on N5 of the reduced flavin adenine dinucleotide (FAD) cofactor, as confirmed using docking and molecular dynamics simulations.     

The Bacterial [Fe]‐Hydrogenase Paralog HmdII Uses Tetrahydrofolate Derivatives as Substrates

[Fe]‐hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl‐tetrahydromethanopterin (methenyl‐H₄MPT⁺) with H₂. H₄MPT is a C1‐carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA–G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron–guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H₄MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1‐carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog.     

Identification and molecular characterization of a novel flavin-free NADPH preferred azoreductase encoded by <Emphasis Type="Italic">azoB</Emphasis> in <Emphasis Type="Italic">Pigmentiphaga kullae</Emphasis> K24

Background  

Microbial degradation of azo dyes is commonly initiated by the reduction of the azo bond(s) by a group of NADH or NADPH dependant azoreductases with many requiring flavin as a cofactor. In this study, we report the identification of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24.     

Enantiocomplementary Epoxidation Reactions Catalyzed by an Engineered Cofactor‐Independent Non‐natural Peroxygenase

Peroxygenases are heme‐dependent enzymes that use peroxide‐borne oxygen to catalyze a wide range of oxyfunctionalization reactions. Herein, we report the engineering of an unusual cofactor‐independent peroxygenase based on a promiscuous tautomerase that accepts different hydroperoxides (t‐BuOOH and H₂O₂) to accomplish enantiocomplementary epoxidations of various α,β‐unsaturated aldehydes (citral and substituted cinnamaldehydes), providing access to both enantiomers of the corresponding α,β‐epoxy‐aldehydes. High conversions (up to 98 %), high enantioselectivity (up to 98 % ee), and good product yields (50–80 %) were achieved. The reactions likely proceed via a reactive enzyme‐bound iminium ion intermediate, allowing tweaking of the enzyme's activity and selectivity by protein engineering. Our results underscore the potential of catalytic promiscuity for the engineering of new cofactor‐independent oxidative enzymes.     

The Cofactor of Tetrachloroethene Reductive Dehalogenase of Dehalospirillum multivorans Is Norpseudo‐B12, a New Type of a Natural Corrinoid

The corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans was isolated in its Coβ‐cyano form. This cofactor represents the main corrinoid found in D. multivorans cells. Analysis of the isolated cyano‐corrinoid by a combination of HPLC and UV/VIS‐absorbance spectroscopy revealed it to be nonidentical to a variety of known natural B₁₂ derivatives. From high‐resolution mass‐spectrometric analysis, the molecular formula of the corrinoid isolated from D. multivorans could be deduced as C₅₈H₈₁CoN₁₇O₁₄P. The sample of the novel corrinoid from D. multivorans was further analyzed by UV/VIS, CD, and one‐ and two‐dimensional ¹H‐, ¹³C‐, and ¹⁵N‐NMR spectroscopy, which indicated its structure to be closely related to that of pseudovitamin B₁₂ (Coβ‐cyano‐7″‐adeninylcobamide). By the same means, the corrinoid could be shown to differ from pseudovitamin B₁₂ only by the lack of the methyl group attached to carbon 176, and, therefore, it was named norpseudovitamin B₁₂ (or, more precisely, 176‐norpseudovitamin B₁₂). Norpseudovitamin B₁₂ represents the first example of a ‘complete’ B₁₂‐cofactor that lacks one of the methyl groups of the cobamide moiety, indicating that the B₁₂‐biosynthetic pathway in D. multivorans differs from that of other organisms. X‐Ray crystal‐structures were determined for norpseudovitamin B₁₂ from D. multivorans and the analogues pseudovitamin B₁₂ and factor A (Coβ‐cyano‐7″‐[2‐methyl]adeninylcobamide). These first accurate crystal structures of complete corrinoids with an adeninyl pseudonucleotide confirmed the expected coordination properties around Co and corroborated the close conformational similarity of the nucleotide moieties of norpseudovitamin B₁₂ and its two homologues.     

Formation of the Δ18,19 Double Bond and Bis(spiroacetal) in Salinomycin Is Atypically Catalyzed by SlnM,a Methyltransferase‐like Enzyme

Salinomycin is a widely used polyether coccidiostat and was recently found to have antitumor activities. However, the mechanism of its biosynthesis remained largely speculative until now. Reported herein is the identification of an unprecedented function of SlnM, homologous to O‐methyltransferases, by correlating its activity with the formation of the Δ^18,19 double bond and bis(spiroacetal). Detailed in vivo and in vitro investigations revealed that SlnM, using positively charged S‐adenosylmethionine (SAM) or sinefungin as the cofactor, catalyzed the spirocyclization‐coupled dehydration of C19 in a highly atypical fashion to yield salinomycin.     

Formation of the Δ18,19 Double Bond and Bis(spiroacetal) in Salinomycin Is Atypically Catalyzed by SlnM,a Methyltransferase‐like Enzyme

Salinomycin is a widely used polyether coccidiostat and was recently found to have antitumor activities. However, the mechanism of its biosynthesis remained largely speculative until now. Reported herein is the identification of an unprecedented function of SlnM, homologous to O‐methyltransferases, by correlating its activity with the formation of the Δ^18,19 double bond and bis(spiroacetal). Detailed in vivo and in vitro investigations revealed that SlnM, using positively charged S‐adenosylmethionine (SAM) or sinefungin as the cofactor, catalyzed the spirocyclization‐coupled dehydration of C19 in a highly atypical fashion to yield salinomycin.