“…The absence of unreacted CoA in the collected fractions and the resuspended lyophilized thioester was confirmed by negative reaction with the DTNB reagent. The optical spectra of the benzoyl‐CoA thioesters showed a π→π * transition around 260 nm corresponding to the adenyl‐moiety of CoA and the thioester linkage, alongside the n→π * transition in the 280–400 nm region corresponding to the substituted phenolic ring (Figures S20 and S21) consistent with literature [28] . The extinction coefficient for the π→π * transition at 260 nm for all the synthesized benzoyl‐CoAs was calculated to be ∼20 000 M −1 cm −1 , agreeing with the published value of 21 000 M −1 cm −1 for benzoyl‐CoA [28] .…”
Section: Resultssupporting
confidence: 87%
“…The optical spectra of the benzoyl-CoA thioesters showed a π!π* transition around 260 nm corresponding to the adenyl-moiety of CoA and the thioester linkage, alongside the n!π* transition in the 280-400 nm region corresponding to the substituted phenolic ring (Figures S20 and S21) consistent with literature. [28] The extinction coefficient for the π!π* transition at 260 nm for all the synthesized benzoyl-CoAs was calculated to be ~20 000 M À 1 cm À 1 , agreeing with the published value of 21 000 M À 1 cm À 1 for benzoyl-CoA. [28] The red shift observed for the n!π* transition ranged from 280-400 nm and was dependent on aromatic ring substituents, and comparison of optical spectra showed that anthraniloyl-CoA displayed a more pronounced red-shift with an absorption maximum at 356 nm as compared to 320 nm for salicyl-CoA (Figure S21).…”
Section: Milligram-scale Coa Thioester Synthesis By Using Ligasesmentioning
confidence: 99%
“…[28] The extinction coefficient for the π!π* transition at 260 nm for all the synthesized benzoyl-CoAs was calculated to be ~20 000 M À 1 cm À 1 , agreeing with the published value of 21 000 M À 1 cm À 1 for benzoyl-CoA. [28] The red shift observed for the n!π* transition ranged from 280-400 nm and was dependent on aromatic ring substituents, and comparison of optical spectra showed that anthraniloyl-CoA displayed a more pronounced red-shift with an absorption maximum at 356 nm as compared to 320 nm for salicyl-CoA (Figure S21). The benzoyl-CoA thioesters were further characterized by highresolution mass spectrometry in negative-ion mode (Figure S22).…”
Section: Milligram-scale Coa Thioester Synthesis By Using Ligasesmentioning
Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants.As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.
“…The absence of unreacted CoA in the collected fractions and the resuspended lyophilized thioester was confirmed by negative reaction with the DTNB reagent. The optical spectra of the benzoyl‐CoA thioesters showed a π→π * transition around 260 nm corresponding to the adenyl‐moiety of CoA and the thioester linkage, alongside the n→π * transition in the 280–400 nm region corresponding to the substituted phenolic ring (Figures S20 and S21) consistent with literature [28] . The extinction coefficient for the π→π * transition at 260 nm for all the synthesized benzoyl‐CoAs was calculated to be ∼20 000 M −1 cm −1 , agreeing with the published value of 21 000 M −1 cm −1 for benzoyl‐CoA [28] .…”
Section: Resultssupporting
confidence: 87%
“…The optical spectra of the benzoyl-CoA thioesters showed a π!π* transition around 260 nm corresponding to the adenyl-moiety of CoA and the thioester linkage, alongside the n!π* transition in the 280-400 nm region corresponding to the substituted phenolic ring (Figures S20 and S21) consistent with literature. [28] The extinction coefficient for the π!π* transition at 260 nm for all the synthesized benzoyl-CoAs was calculated to be ~20 000 M À 1 cm À 1 , agreeing with the published value of 21 000 M À 1 cm À 1 for benzoyl-CoA. [28] The red shift observed for the n!π* transition ranged from 280-400 nm and was dependent on aromatic ring substituents, and comparison of optical spectra showed that anthraniloyl-CoA displayed a more pronounced red-shift with an absorption maximum at 356 nm as compared to 320 nm for salicyl-CoA (Figure S21).…”
Section: Milligram-scale Coa Thioester Synthesis By Using Ligasesmentioning
confidence: 99%
“…[28] The extinction coefficient for the π!π* transition at 260 nm for all the synthesized benzoyl-CoAs was calculated to be ~20 000 M À 1 cm À 1 , agreeing with the published value of 21 000 M À 1 cm À 1 for benzoyl-CoA. [28] The red shift observed for the n!π* transition ranged from 280-400 nm and was dependent on aromatic ring substituents, and comparison of optical spectra showed that anthraniloyl-CoA displayed a more pronounced red-shift with an absorption maximum at 356 nm as compared to 320 nm for salicyl-CoA (Figure S21). The benzoyl-CoA thioesters were further characterized by highresolution mass spectrometry in negative-ion mode (Figure S22).…”
Section: Milligram-scale Coa Thioester Synthesis By Using Ligasesmentioning
Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants.As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.
“…These factors taken together suggest the p K a s for 4-HBA-CoA binding to D145S or D145A are in the region of 6.5−7.0. This is approximately 1.5−2 units below that found for free 4-HBA-CoA ( , ) and demonstrates how the active site can manipulate electron density at the 4-position of the bound ligand, which is likely a prerequisite for the aromatic substitution reaction occurring at that point. 7 pH dependence of the absorbance of the 388 nm peak due to ionized 4-HBA-CoA on D145S dehalogenase.…”
The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBACoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in λ max , and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's π-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH 2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O -) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with λ maxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pK a for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pK a for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm -1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 µM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.
“…A large window of potentials was made accessible by the use of redox dyes ranging from anthraquinone 1,5-disulfonate to methyl viologen. We also followed reduction of hydroxybenzoyl-CoA that has an absorption peak at 299 nm (Webster et al, 1974). At this wavelength, benzyl viologen has only a weak absorbance.…”
Hydroxybenzoyl-CoA reductase (HBCR) is an iron-sulfur protein that is involved in the metabolism of aromatic compounds. It catalyzes the two-electron reduction of hydroxybenzoyl-CoA to benzoyl-CoA. In the work described here, kinetic schemes were derived for HBCR and for several classes of redox enzymes and redox-activated enzymes. Introduction of the Nernst equation into the rate laws led to the development of novel relationships between the ambient redox potential, the midpoint potential of the enzyme active site, and the kinetic parameter, V/K. By coupling electrochemistry and steady-state kinetics, mechanistic information could be obtained that could not be determined by either method alone. For HBCR, the relationship between the kinetic parameter V/K and the ambient electrochemical potential of the assay mixture was found to be: apparent V/Km = Vmax/(Km(1 + exp[nF/RT(E - E(o)e)])), where n is the number of electrons involved in the redox process, F is the Faraday constant, R is the gas constant, T is the temperature in K, E is the applied potential, and E(o)e is the redox potential of a redox-active catalytic site on the enzyme. Coupling kinetics with electrochemistry yielded the E(o)e (-350 mV vs NHE) for HBCR and maximum values under optimal redox conditions for kcat and kcat/Km (9 s-1 and 1.8 x 10(5) M-1 s-1, respectively). In addition, theory was developed that could distinguish a single two-electron transfer mechanism from one involving two successive one-electron transfers. HBCR was found to be in the latter class. Interestingly, the derived mechanism for HBCR is similar to that of the Birch reduction, the classical organic chemical reaction for reductive dehydroxylation of phenolic compounds. The methodology described here represents a novel approach that should help elucidate the mechanisms of other oxidoreductase and redox-activated enzymes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
