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SIRT5, one of the mammalian sirtuins, specifically recognizes succinyl‐lysine residues on proteins and catalyzes the desuccinylation reaction. In this study, we characterized SIRT5 mutants with hydrophobic amino acid substitutions at Q140 and N141, in addition to the catalytic residue H158, known as an active site residue, by the Michaelis–Menten analysis and X‐ray crystallography. Kinetic analysis showed that the catalytic efficiency (kcat/Km) of the Q140L and N141V mutants decreased to 0.02 times and 0.0038 times that of the wild‐type SIRT5, respectively, with the activity of the N141V mutant becoming comparable to that of the H158M mutant. Our findings indicate that N141 contributes significantly to the desuccinylation reaction. [ABSTRACT FROM AUTHOR]

The nature of air-inactivation of the formate dehydrogenase FdsDABG from Cupriavidus necator has been investigated. It is found that superoxide, generated in the reaction of reduced enzyme with oxygen, is responsible for the loss of activity and that superoxide dismutase protects the enzyme from air-inactivation. Inhibition appears to be due to the reaction of superoxide with the catalytically essential MoS group of the enzyme's molybdenum center in such a way that generates sulfite. SYNOPSIS: Superoxide generated in the reaction of reduced formate dehydrogenase FdsDABG from Cupriavidus necator with O2 is found to be responsible for the loss of activity. Catalytic amounts of superoxide dismutase are found to protect FdsDABG just as well as more generally used stabilizing inhibitors such as nitrate.

MnxCo3‐x[Co(CN)6]2 Prussian blue analogues were synthesized with Mn : Co ratios in the range from 7 : 10 to 1 : 11 and pyrolyzed at 600 °C to obtain a highly nitrogen‐ and oxygen‐functionalized carbon matrix with embedded reduced Mn and Co species. These catalyst precursors were applied in the CO hydrogenation to higher alcohols at 260 °C and a pressure of 60 bar using a H2/CO ratio of 1. Metallic Co0 formed during pyrolysis was partially transformed into Co2C under reaction conditions. With increasing Co content CO conversion increased up to 10.6 % reaching a total alcohol selectivity of 19 %. Gas chromatograms revealed the expected formation of primary short‐chain alcohols, but also of secondary alcohols, acetic acid and propionaldehyde indicating olefin hydration, carbonylation and hydroformylation as reaction pathways, respectively. The obtained hydrocarbon fractions had a very high olefinicity, which is beneficial for both olefin hydration to secondary alcohols catalyzed by adsorbed carboxylic acids and for hydroformylation. Whereas the carbide‐based reaction pathway and the reductive hydroformylation are assumed to occur at the Co2C/Co0 interface, carbonylation is presumably catalyzed by an additional Co‐based active site. Thus, a unique class of multifunctional catalysts was obtained with highly promising properties bridging the gap between heterogeneous and homogeneous catalysis. [ABSTRACT FROM AUTHOR]

Oxidoreductases catalyze essential redox reactions, and many require a diffusible cofactor for electron transport, such as NAD(H). Non-canonical cofactor analogs have been explored as a means to create enzymatic reactions that operate orthogonally to existing metabolism. Here, we aimed to engineer the formate dehydrogenase from Candid boidinii (CbFDH) for activity with the non-canonical cofactor nicotinamide adenine dinucleotide 3′-phosphate (3′-NADP(H)). We used PyRosetta, the Cofactor Specificity Reversal Structural Analysis and Library Design (CSR-SALAD), and structure-guided saturation mutagenesis to identify mutations that enable CbFDH to use 3′-NADP+. Two single mutants, D195A and D195G, had the highest activities with 3′-NADP+, while the double mutant D195G/Y196S exhibited the highest cofactor selectivity reversal behavior. Steady state kinetic analyses were performed; the D195A mutant exhibited the highest KTS value with 3′-NADP+. This work compares the utility of computational approaches for cofactor specificity engineering while demonstrating the engineering of an important enzyme for novel non-canonical cofactor selectivity. [ABSTRACT FROM AUTHOR]

β- N -acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii , formerly named V. harveyi , (Vh GlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by Vh GlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N -acetyl- β -D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a K d of 99.1 ± 1 μM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k 2) of 53.3 ± 1 s−1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k 3) of 14.6 s−1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for Vh GlcNAcase. • Structure-based phylogenetic analysis placed Vh GlcNAcase from Vibrio campbellii in Clade A-I of GlcNAcases' phylogenetic tree. • Pre-steady-state and steady-state kinetic analysis of Vh GlcNAcase with 4MU-GlcNAc revealed a distinctive three-step "ping-pong" mechanism. [ABSTRACT FROM AUTHOR]

Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD+ with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD+ to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN+). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD+ decreased significantly except for the T11 mutants but changed slightly for NMN+ except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN+ by 8.7-fold, concomitant with a significant decrease in NAD+ by 1.4 × 104-fold, resulting in 9.6 × 104-fold cofactor specificity switch toward NMN+ over NAD+. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD+ and NMN+, thereby affecting their activity. A70K variant sterically blocks the binding with NAD+, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN+, and enhances the catalytic efficiency for NMN+. [ABSTRACT FROM AUTHOR]

SIRT5, one of the mammalian sirtuins, specifically recognizes succinyl-lysine residues on proteins and catalyzes the desuccinylation reaction. In this study, we characterized SIRT5 mutants with hydrophobic amino acid substitutions at Q140 and N141, in addition to the catalytic residue H158, known as an active site residue, by the Michaelis-Menten analysis and X-ray crystallography. Kinetic analysis showed that the catalytic efficiency (k cat /K m ) of the Q140L and N141V mutants decreased to 0.02 times and 0.0038 times that of the wild-type SIRT5, respectively, with the activity of the N141V mutant becoming comparable to that of the H158M mutant. Our findings indicate that N141 contributes significantly to the desuccinylation reaction., (© 2024 Federation of European Biochemical Societies.)

Agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine, is a member of the GCN5-related N-acetyltransferase family. So far, knowledge of the physiological roles of AgmNAT in insects is limited. Here, we identified one gene encoding protein homologous to that of Drosophila AgmNAT using sequence information from an activity-verified Drosophila AgmNAT in a BLAST search of the Bactrocera dorsalis genome. We expressed and purified B. dorsalis AgmNAT in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Our application of the screening strategy to BdorAgmNAT led to the identification of agmatine as the best amine substrate for this enzyme, with the highest k cat /K m value. We successfully obtained a BdorAgmNAT knockout strain based on a wild-type strain (WT) using the CRISPR/Cas9 technique. The ovary development of the BdorAgmNAT knockout mutants was delayed for 10 days compared with the WT specimens. Moreover, mutants had a much smaller mature ovary size and laid far fewer eggs than WT. Loss of function of BdorAgmNAT caused by RNAi with mature WT females did not affect their fecundity. These findings indicate that BdorAgmNAT is critical for oogenesis. Our data provide the first evidence for AgmNAT in regulating ovary development. [Display omitted] • Enzymatic properties of the purified recombinant BdorAgmNAT was analyzed. • We generated a BdorAgmNAT knockout mutant using the CRISPR/Cas9 system. • BdorAgmNAT gene is critical for ovary development. [ABSTRACT FROM AUTHOR]

l-Thioproline (l-thiazolidine-4-carboxylate, l-T4C) is a cyclic sulfur-containing analog of l-proline found in multiple kingdoms of life. The oxidation of l-T4C leads to l-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of l-T4C to l-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and l-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with l-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant l-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M−1 s−1) than PYCR1 (13.7 M−1 s−1). Interestingly, no activity was observed with either l-Pro or the analog dl-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize l-T4C as a substrate. Inhibition kinetics show that l-Pro is a competitive inhibitor of PYCR1 K IC app = 15.7 mM with respect to l-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that l-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of l-T4C. [ABSTRACT FROM AUTHOR]

Dominant mutations in p97/VCP (valosin-containing protein) cause a rare multisystem degenerative disease with varied phenotypes that include inclusion body myopathy, Paget's disease of bone, frontotemporal dementia, and amyotrophic lateral sclerosis. p97 disease mutants have altered N-domain conformations, elevated ATPase activity, and altered cofactor association. We have now discovered a previously unidentified disease-relevant functional property of p97 by identifying how the cofactors p37 and p47 regulate p97 ATPase activity. We define p37 as, to our knowledge, the first known p97-activating cofactor, which enhances the catalytic efficiency (kcat/Km) of p97 by 11-fold. Whereas both p37 and p47 decrease the Km of ATP in p97, p37 increases the kcat of p97. In contrast, regulation by p47 is biphasic, with decreased kcat at low levels but increased kcat at higher levels. By deleting a region of p47 that lacks homology to p37 (amino acids 69-92), we changed p47 from an inhibitory cofactor to an activating cofactor, similar to p37. Our data suggest that cofactors regulate p97 ATPase activity by binding to the N domain. Induced conformation changes affect ADP/ATP binding at the D1 domain, which in turn controls ATPase cycling. Most importantly, we found that the D2 domain of disease mutants failed to be activated by p37 or p47. Our results show that cofactors play a critical role in controlling p97 ATPase activity, and suggest that lack of cofactor-regulated communication may contribute to p97-associated disease pathogenesis.

Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD+ with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD+ to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN+). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD+ decreased significantly except for the T11 mutants but changed slightly for NMN+ except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN+ by 8.7-fold, concomitant with a significant decrease in NAD+ by 1.4 × 104-fold, resulting in 9.6 × 104-fold cofactor specificity switch toward NMN+ over NAD+. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD+ and NMN+, thereby affecting their activity. A70K variant sterically blocks the binding with NAD+, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN+, and enhances the catalytic efficiency for NMN+.

The surge in vancomycin-resistant enterococci (VRE) strains poses a serious threat to public and clinical health. VRE strains are the leading cause of multi-drug resistant enterococcal infections and are commonly acquired from medical devices. Therefore, it is essential to discover new antibacterial targets and drugs for this pathogen. Alanine racemase could be a valuable drug target due to its crucial role in E. faecium survival. Alr from E. faecium (EF_Alr) was heterologously produced and purified from E. coli., and the steady-state kinetic constants were determined at different pH values. Using a coupled reaction with L-alanine dehydrogenase, rate of production of NADH was measured at 340 nm to observe EF_Alr activity in the D- to L-alanine direction. The highest catalytic efficiency, 8.61 ± 0.5 s-1 mM-1, was found at pH 9. Additionally, the tentative active site residues, Lys40 and Tyr268, for the alanine racemization reaction were assigned by homology modeling and sequence comparison studies. Using UCSF Chimera, the structure of the EF_Alr homology model was superimposed and compared to the crystal structure of Alr from E. faecalis. [ABSTRACT FROM AUTHOR]

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (k cat/K m) of D1 ATP hydrolysis 280-fold, by increasing k cat 7-fold and decreasing K m about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97. © 2014 Elsevier Ltd.

The final arbiter of enzyme mechanism is the ability to establish and test a kinetic mechanism. Isotope effects play a major role in expanding the scope and insight derived from the Michaelis-Menten equation. The integration of isotope effects into the formalism of the Michaelis-Menten equation began in the 1970s and has continued until the present. This review discusses a family of eukaryotic copper proteins, including dopamine β-monooxygenase, tyramine β-monooxygenase and peptidylglycine α-amidating enzyme, which are responsible for the synthesis of neuroactive compounds, norepinephrine, octopamine and C-terminally carboxamidated peptides, respectively. The review highlights the results of studies showing how combining kinetic isotope effects with initial rate parameters permits the evaluation of: (a) the order of substrate binding to multisubstrate enzymes; (b) the magnitude of individual rate constants in complex, multistep reactions; (c) the identification of chemical intermediates; and (d) the role of nonclassical (tunnelling) behaviour in C-H activation.

• Two-dimensional gas chromatography was applied to elucidate the reaction network. • Same chain growth probabilities for alcohols, olefins and hydrocarbons. • Olefins are primary products, alkanes are formed consecutively. • Alcohol selectivity decreases with increasing conversion of CO. • Increased availability of hydrogen deteriorates chain growth and CO insertion. The kinetics of higher alcohol synthesis was investigated using a hydrotalcite-derived Co-Cu-based catalyst aiming at a deeper understanding of the complex reaction network. At steady state similar chain growth probabilities of about 0.4 according to the Anderson-Schulz-Flory distribution were observed for alcohols, hydrocarbons and olefins indicating common intermediates. Alkanes were found to be formed consecutively from primarily formed olefins. The observed decrease of the selectivities to alcohols with increasing CO conversion at higher temperatures and higher residence times is ascribed to an increased availability of adsorbed atomic hydrogen, which decreases the saturated coverage of CO-derived C x H y O z species favoring hydrocarbon formation. Correspondingly, reaction orders of 0 and 0.8 for CO and H 2 , respectively, were derived based on a power-law approach including an apparent activation energy of 140 kJ mol−1. A reaction network based on the CO insertion factor was established, in which the competing reactions β -hydrogen elimination, chain growth and CO insertion proceed from common adsorbed C x H y intermediates. Selective higher alcohol formation was favored at low temperatures and short residence times, high pressures and a moderate H 2 :CO ratio of 1 requiring a compromise between conversion and selectivity. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase I from Trichoderma reesei (TrCel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of TrCel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of TrCel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of TrCel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of TrCel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake (kon for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel. [ABSTRACT FROM AUTHOR]

A method has been presented for solving the inverse problem of chemical kinetics based on steady-state experimental data with different initial conditions (multiexperiments) taking into account measurement errors in an open nonisothermal gradientless reactor. Examples of using the method to determine the intervals of the physical values of rate constants for the stages of multistage nonlinear reactions have been given. The effect of measurement errors on the accuracy and stability of the method has been evaluated. [ABSTRACT FROM AUTHOR]

Quantitative evaluation of the antioxidant capacity of foods is of great significance for estimating food’s nutritional value and preventing oxidative changes in food. Herein, we demonstrated an easy and selective colorimetric method for the total antioxidant capacity (TAC) assay based on 3,3’,5,5’-tetramethyl-benzidine (TMB), hydrogen peroxide (H2O2) and synthetic Lycium barbarum polysaccharide-iron (III) chelate (LBPIC) with high peroxidase (POD)-like activity. The results of steady-state kinetics study showed that the Km values of LBPIC toward H2O2 and TMB were 5.54 mM and 0.16 mM, respectively. The detection parameters were optimized, and the linear interval and limit of detection (LOD) were determined to be 2–100 μM and 1.51 μM, respectively. Additionally, a subsequent study of the determination of TAC in six commercial fruit and vegetable beverages using the established method was successfully carried out. The results implied an expanded application of polysaccharide-iron (III) chelates with enzymatic activity in food antioxidant analysis and other biosensing fields.

F420H2:NADP+ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP+ to NADPH by transferring a hydride from the reduced F420 cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon wtFno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP+ and the F420 precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of wtFno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased kcat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs420 versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s−1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH2 to NADP+. Therefore, Fno is a dynamic enzyme that regulates NADPH production.

• Epigenetically modified 6 mA and its intermediate hypoxanthine (I) partially inhibited DNA replication. • dCMP incorporation opposite I can be considered as correct incorporation. • 6 mA and I reduced correct incorporation and next-base extension efficiencies. • 6 mA and I reduced the burst incorporation rate and increased dNTP dissociation constant. • 6 mA and I reduced the binding affinity of hPol η to DNA in both binary and ternary complexes. N 6-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication remain elusive. In this work, we used Y-family human DNA polymerase η as a model to investigate the kinetics of bypass of 6mA by hPol η. We found 6mA and its intermediate hypoxanthine (I) on template partially inhibited DNA replication by hPol η. dTMP incorporation opposite 6mA and dCMP incorporation opposite I can be considered as correct incorporation. However, both 6mA and I reduced correct incorporation efficiency, next-base extension efficiency, and the priority in extension beyond correct base pair. Both dTMP incorporation opposite 6mA and dCTP opposite I showed fast burst phases. However, 6mA and I reduced the burst incorporation rates (k pol) and increased the dissociation constant (K d,dNTP), compared with that of dTMP incorporation opposite unmodified A. Biophysical binding assays revealed that both 6mA and I on template reduced the binding affinity of hPol η to DNA in binary or ternary complex compared with unmodified A. All the results explain the inhibition effects of 6mA and I on DNA replication by hPol η, providing new insight in the effects of epigenetically modified 6mA on human DNA replication. [ABSTRACT FROM AUTHOR]

Abstract The binding energy of enzyme and substrate is used to lower the activation energy for the catalytic reaction. 3α-HSD/CR uses remote binding interactions to accelerate the reaction of androsterone with NAD+. Here, we examine the enthalpic and entropic components of the remote binding energy in the 3α-HSD/CR-catalyzed reaction of NAD+ with androsterone versus the substrate analogs, 2-decalol and cyclohexanol, by analyzing the temperature-dependent kinetic parameters through steady-state kinetics. The effects of temperature on k cat /K m for 3α-HSD/CR acting on androsterone, 2-decalol, and cyclohexanol show the reactions are entropically favorable but enthalpically unfavorable. Thermodynamic analysis from the temperature-dependent values of K m and k cat shows the binding of the E-NAD+ complex with either 2-decalol or cyclohexanol to form the ternary complex is endothermic and entropy-driven, and the subsequent conversion to the transition state is both enthalpically and entropically unfavorable. Hence, solvation entropy may play an important role in the binding process through both the desolvation of the solute molecules and the release of bound water molecules from the active site into bulk solvent. As compared to the thermodynamic parameters of 3α-HSD/CR acting on cyclohexanol, the hydrophobic interaction of the B-ring of steroids with the active site of 3α-HSD/CR contributes to catalysis by increasing exclusively the entropy of activation (ΔTΔS‡ = 1.8 kcal/mol), while the BCD-ring of androsterone significantly lowers ΔΔH‡ by 10.4 kcal/mol with a slight entropic penalty of −1.9 kcal/mol. Therefore, the remote non-reacting sites of androsterone may induce a conformational change of the substrate binding loop with an entropic cost for better interaction with the transition state to decrease the enthalpy of activation, significantly increasing catalytic efficiency. Graphical abstract Image 1 Highlights •. 3α-HSD/CR catalyzed reaction is entropy-driven. •. Binding of E-NAD binary complex with 2-decalol or cyclohexanol is endothermic and entropy-driven. •. Formation of the transition state from ternary complex is both enthalpically and entropically unfavorable. •. The hydrophobic interactions of B-ring of steroids contribute to catalysis by increasing TΔS‡. •. The BCD-ring of androsterone lowering ΔH‡ results in the significant increases in kcat/Km. [ABSTRACT FROM AUTHOR]

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro‐ and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so‐called Prx6‐type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady‐state kinetics with tert‐butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four‐ to six‐fold in vitro. Stopped‐flow kinetics with reduced PfPrx6WT, PfPrx6C128A, and PfPrx6H39Y revealed a preference for H2O2 as an oxidant with second order rate constants for H2O2 and tBuOOH around 2.5 × 107 M−1 s−1 and 3 × 106 M−1 s−1, respectively. Differences between the oxidation kinetics of PfPrx6WT, PfPrx6C128A, and PfPrx6H39Y were observed during a slower second‐reaction phase. Our kinetic data support the interpretation that the reductive half‐reaction is the rate‐limiting step for PfPrx6 catalysis in steady‐state measurements. Whether the increased activity of PfPrx6H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6‐type enzymes is non‐essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain‐of‐function mutant PfPrx6H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues. [ABSTRACT FROM AUTHOR]

Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X‐ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH‐binding site (G‐site) and a hydrophobic co‐substrate‐binding site (H‐site). At elevated H2O2 concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H‐site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox‐regulated enzyme. PDB Code(s): 6F05; 6F01; 6EZY [ABSTRACT FROM AUTHOR]

Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes. While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems, the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts. Only a few commercial microfluidic systems for enzyme analysis are currently available, and the transformation of numerous published prototypes into commercial platforms is still to be streamlined. This review presents the state-of-the-art, recent trends, and perspectives in applying microfluidic tools in the functional and structural analysis of biocatalysts. We discuss the advantages and disadvantages of available technologies, their reproducibility and robustness, and readiness for routine laboratory use. We also highlight the unexplored potential of microfluidics to leverage the power of machine learning for biocatalyst development. [Display omitted] • Biocatalysis depends on the identification and characterization of novel enzymes. • Efficient tools for high-throughput characterization of biocatalysts are needed. • The rapidly developing microfluidics shows considerable application potential. • Translation of microfluidic prototypes to ready-to-use instruments is underdeveloped. • Microfluidics has great potential to provide high-quality data for machine learning. [ABSTRACT FROM AUTHOR]

Abnormal H2O2 and cholesterol levels are closely related to many diseases. This work reports a facile process for the synthesis of oxidized glutathione (GSSG)‐modified MoS2 nanosheets (MoS2‐GSSG NSs). The biocompatible MoS2‐GSSG NSs have good dispersibility and high affinity to the substrate 3,3′,5,5′‐tetramethylbenzidine (TMB), which is beneficial for improving peroxidase‐like catalytic activity of MoS2. The high peroxidase‐like activity of MoS2‐GSSG NSs was applied as a robust nanoplatform for low‐cost, rapid, and highly effective colorimetric detection of H2O2 and total/free cholesterol. Moreover, the peroxidase‐like catalytic mechanism was studied by the steady‐state kinetics method. The catalytic activity was remarkably high at a wide range of pH (2.4–7.0) and temperature values (25–70 °C). The cholesterol was catalyzed by cholesterol oxidase (ChOx) in the presence of O2 to generate H2O2, which oxidized TMB to generate a blue‐colored product (oxTMB) under the catalysis of MoS2‐GSSG NSs. The detection limit (DL) of total cholesterol and H2O2 was as low as 5.36 and 0.51 μm, respectively. The linear ranges for detecting cholesterol and H2O2 were from 5.36 to 800 μm and from 0.51 to 50 μm, respectively. This method was also successfully applied to the detection of cholesterol in serum. The detection concentration of total cholesterol was consistent with that of the value detected by the blood biochemical method used in the clinic. Oxidized glutathione (GSSG)‐modified MoS2 nanosheets (MoS2‐GSSG NSs) that have good biocompatibility and dispersibility and high peroxidase‐like catalytic activity over a wide pH value and temperature range were simply constructed (see scheme). The MoS2‐GSSG NSs were applied as a robust nanoplatform for low‐cost, rapid, and highly effective colorimetric detection of H2O2 and cholesterol. [ABSTRACT FROM AUTHOR]

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting coordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the coordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies. [ABSTRACT FROM AUTHOR]

The Ca 2+ -dependent deamidation and transamidation activities of transglutaminase 2 (TG2) are important to numerous physiological and pathological processes. Herein, we have examined the steady-state kinetics and 15 (V/K) kinetic isotope effects (KIEs) for the TG2-catalyzed deamidation and transamidation of N -Benzyloxycarbonyl- l -Glutaminylglycine (Z-Gln-Gly) using putrescine as the acyl acceptor substrate. Kinetic parameters determined from initial velocity plots are consistent with previously proposed mechanisms. Significant differences in the 15 (V/K) KIEs on NH 3 release determined for the deamidation (0.2%) and the transamidation (2.3%) of Z-Gln-Gly suggest the rate-limiting steps of TG2 active site acylation are dependent on the presence of the acyl acceptor. We propose a plausible mechanistic explanation where substrate-induced conformational changes may play a role in promoting catalysis. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatases (PTPases) are a prominent focus of drug design studies because of their roles in homeostasis and disorders of metabolism. These studies have met with little success because (1) virtually all inhibitors hitherto exhibit only competitive behavior and (2) a consensus sequence H/V-C-X-R-S/T characterizes the active sites of PTPases, leading to low specificity of active site directed inhibitors. With protein tyrosine phosphatase-1B (PTP1B) identifed as the target enzyme of the vanadyl (VO) chelate bis(acetylacetonato)oxidovanadium(IV) [VO(acac)] in 3T3-L1 adipocytes [Ou et al. J Biol Inorg Chem 10: 874-886, 2005], we compared the inhibition of PTP1B by VO(acac) with other VO-chelates, namely, bis(2-ethyl-maltolato)oxidovanadium(IV) [VO(Et-malto)] and bis(3-hydroxy-2-methyl-4(1H)pyridinonato)oxidovanadium(IV) [VO(mpp)] under steady-state conditions, using the soluble portion of the recombinant human enzyme (residues 1-321). Our results differed from those of previous investigations because we compared inhibition in the presence of the nonspecific substrate p-nitrophenylphosphate and the phosphotyrosine-containing undecapeptide DADEpYLIPQQG mimicking residues 988-998 of the epidermal growth factor receptor, a relevant, natural substrate. While VO(Et-malto) acts only as a noncompetitive inhibitor in the presence of either subtrate, VO(acac) exhibits classical uncompetitive inhibition in the presence of DADEpYLIPQQG but only apparent competitive inhibition with p-nitrophenylphosphate as substrate. Because uncompetitive inhibitors are more potent pharmacologically than competitive inhibitors, structural characterization of the site of uncompetitive binding of VO(acac) may provide a new direction for design of inhibitors for therapeutic purposes. Our results suggest also that the true behavior of other inhibitors may have been masked when assayed with only p-nitrophenylphosphate as substrate. [ABSTRACT FROM AUTHOR]

The catalytic cycle of the cytochromes P450 (CYP) requires two electrons from a protein redox partner and two protons from water to generate the main catalytic intermediate, a ferryl-oxo complex with π-cation on the heme porphyrin ring, termed Compound 1. The protonation steps are at least partially rate-limiting, therefore the steady-state rates of P450 catalysis are usually slower in deuterated solvent (D 2 O) by a factor of 1.5–3. However, in several P450 systems a pronounced inverse kinetic solvent isotope effect (KSIE ∼0.4–0.7) is observed, where the reaction is faster in D 2 O. This raises an important mechanistic question: Is this inverse solvent isotope effect compatible with Compound 1 catalyzed reactions, or is it indicative of another catalytic intermediate being involved? In this communication we use exhaustive numerical modeling of the P450 steady-state kinetics to demonstrate that a significant inverse KSIE cannot be obtained for a pure Compound 1 driven catalytic cycle of P450. Rather, an alternative, protonation independent, catalytic intermediate needs to be introduced. This result is applicable to the broad spectrum of P450s in nature, but as an example we use the extensively documented inverse isotope effect in the human steroid biosynthetic P450 CYP17A1 where the involvement of a heme peroxo anion intermediate has been characterized. Based on this analysis, we show that the observation of an inverse KSIE can be used as a general mechanistic probe for reaction cycle intermediates in the cytochromes P450. Numerical modeling of cytochrome P450 steady-state kinetics demonstrates that the product formation via Compound 1 pathways always shows a normal kinetic solvent isotope effect (KSIE), in agreement with all available experimental data. Contrary, the inverse KSIE observed for the lyase reaction in CYP17A1 requires the alternative pathway via peroxo-ferric intermediate. [Display omitted] • Modeling of cytochrome P450 kinetics demonstrates KSIE is a sensitive probe of catalytic mechanism. • Compound 1 mediated catalysis always shows a normal (>1) kinetic solvent isotope effect (KSIE). • An inverse KSIE (<1) can be obtained only by protonation independent pathway. • Catalysis via the peroxoanion intermediate always yields an inverse KSIE. [ABSTRACT FROM AUTHOR]

Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography–mass spectrometry (GC–MS). In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity. • We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs. • We validate the method by direct comparison with established assays. The agreement of kcat/KM among methods makes this adaptation optimal for rapid evaluation of TPSs. • We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries.

Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis.

The world of medicinal therapies has been historically, and remains to be, dominated by the use of elegant organic molecular structures. Now, a novel medical treatment is emerging based on CeO2 nano-crystals that are discrete clusters of a few hundred atoms. This development is generating a great deal of exciting and promising research activity, as evidenced by this Special Issue of Biomolecules. In this paper, we provide both a steady-state and time-dependent mathematical description of a sequence of reactions: superoxide generation, superoxide dismutase, and hydrogen peroxide catalase and ceria regeneration. This sequence describes the reactive oxygen species (ROS); superoxide, O2−, molecular oxygen, O2, hydroxide ion OH− and hydrogen peroxide, H2O2, interacting with the Ce3+, and Ce4+ surface cations of nanoparticle ceria, CeO2. Particular emphasis is placed on the predicted time-dependent role of the Ce3+/Ce4+ ratio within the crystal. The net reaction is succinctly described as: H2O2 + 2O2− + 2H+ → 2H2O + 2O2. The chemical equations and mathematical treatment appears to align well with several critical in vivo observations such as; direct and specific superoxide dismutase (SOD), ROS control, catalytic regeneration, ceria self-regulation and self-limiting behavior. However, in contrast to experimental observations, the model predicts that the 4+ ceric ion state is the key SOD agent. Future work is suggested based on these calculations.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD + to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in k cat , and 37-, 1.9 × 10 6 -, and 1.8 × 10 6 -fold in k cat /K B , respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD + based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The k cat /K B value results in Δ G ‡ of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in k cat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis, resulting in the increases in k cat and k cat /K B . [ABSTRACT FROM AUTHOR]

O 6 -Methylguanine ( O 6 -MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O 6 -MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O 6 -MeG by gp90 exo − . O 6 -MeG partially inhibited full-length extension by gp90 exo − . O 6 -MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo − extends beyond T: O 6 -MeG 2-fold more efficiently than C: O 6 -MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O 6 -MeG show fast burst phases. The pre-steady-state incorporation efficiency ( k pol / K d,dNTP ) is decreased in the order of dCTP:G > dTTP: O 6 -MeG > dCTP: O 6 -MeG. The presence of O 6 -MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg 2+ . Misincorporation of dTTP opposite O 6 -MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O 6 -MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O 6 -MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1. [ABSTRACT FROM AUTHOR]

Background Hydrolysis of β-lactam antibiotic by β-lactamase is the most common mechanism of β-lactam resistance in clinical isolates. Timely detection and characterization of β-lactamases are therefore of utmost biomedical importance. Conventional spectrophotometric method is time-consuming and cannot provide thermodynamic information on β-lactamases. Methods A new assay was developed for the study of β-lactamase activity in protein solutions (Metallo-β-lactamase L1) and in clinical bacterial cells, based on heat-flow changes derived from enzymatic hydrolysis of β-lactams using isothermal titration calorimetry. Results (1) The thermokinetic parameters of three antibiotics (penicillin G, cefazolin and imipenem) and the inhibition constant of an azolylthioacetamide inhibitor were determined using the calorimetric assay. The results from the calorimetric assays were consistent with the data from the spectrophotometric assay. (2) The values of heat change in the calorimetric assay using two clinical Escherichia coli strains correlated well with their antibiotic susceptibility results from the broth dilution experiment. The subtypes of β-lactamase were also determined in the calorimetric assay. Conclusions The ITC assay is a reliable and fast method to study β-lactamase enzyme kinetics and inhibition. It can also provide thermodynamic information on antibiotic hydrolysis, which has been taken advantage of in this work to study β-lactamase activity in two clinical Escherichia coli isolates. General significance As the first calorimetric study of β-lactamase activity, it may provide a new assay to assist biomedical validation of new β-lactamase inhibitors, and also has potential applications on rapid antibiotic susceptibility testing and screening β-lactamase producing bacteria. [ABSTRACT FROM AUTHOR]

Proteases are a class of enzymes that lower the activation energy for the cleavage of the peptide bonds by polarizing the carbonyl group. The catalytic mechanism of proteases is characterized by the formation and the dissociation of a tetrahedral acyl-intermediate. The rate-limiting step in catalysis is either the acylation process (leading to the release of the newly formed $$ {-}{\text{NH}}_{3}^{ + } $$ terminal) or the subsequent deacylation step (leading to the release of the newly formed -COO terminal). As a case, the detailed kinetic analysis for the hydrolysis of the chromogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (wherein Mu is the morpholinocarbonyl protecting group and AMC is the 7-amino-4-methylcoumarin chromophoric group) by the prostate-specific antigen (PSA) is reported here. The pH dependence of the catalytic parameters clearly indicates the existence of protonation/deprotonation processes involving (at least) two ionizing groups in the proximity of the active site. In view of the physio-pathological relevance of PSA in prostate diseases (including cancer), the detailed analysis of the catalytic parameters opens new scenarios for the design of selective inhibitors, which might influence the 'in vivo' activity of this protease. [ABSTRACT FROM AUTHOR]

Aiming to monitor hydrogen peroxide and oxidizing ions in aqueous circumstance, functionalized attapulgite [i.e. gold‐modified attapulgite nanocomposites (Au/ATP NCs)] as peroxidase mimics were prepared by loading β‐cysteamine‐capped gold nanoparticles onto attapulgite with the use of electrostatic interactions. As‐prepared Au/ATP NCs were used for the detection of H2 O2. The linear range and detection limit for H2 O2 were determined by quantitative experiments emerging excellent stability and recyclability. The peroxidase‐like activity of Au/ATP NCs could be expanded for the detection of some oxidative ions (Fe3+ and Ag+) was also been discovered. Based on the absorption spectrum and steady‐state kinetics, the mechanism for peroxidase mimic reaction is investigated. This work represents the first example of multitarget detection for molecule and cations (H2 O2, Fe3+ and Ag+) using Au/ATP NCs as peroxidase enzyme mimics and holds a promise for future biomedical and analytical applications. [ABSTRACT FROM AUTHOR]

As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-20-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of Pseudomonas aeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G. Gp90 D234A could extend beyond C:8-oxoG or A:8-oxoG base pairs with the same efficiency. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG showed fast burst phases. The burst of incorporation efficiency (kpol/Kd,dNTP) is decreased as dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. The presence of 8-oxoG in DNA does not affect its binding to Gp90 D234A in a binary complex but it does affect it in a ternary complex with dNTP and Mg2+, and dATP misincorporation opposite 8-oxoG further weakens the binding of Gp90 D234A to DNA. This study reveals Gp90 D234A can bypass 8-oxoG in an error-free manner, providing further understanding in DNA replication encountering oxidation lesion for P.aeruginosa phage PaP1. [ABSTRACT FROM AUTHOR]

Brugia malayi ( B. malayi ) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase ( Bm DHFR). A His 6 -tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity k cat was found to be 1.4 ± 0.1 s −1 , the Michaelis Menten constant K M for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant K D for NADPH 25 ± 24 nM. For BmDHFR, IC 50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR. [ABSTRACT FROM AUTHOR]

Enzymes require flexible regions to adopt multiple conformations during catalysis. The mobile regions of enzymes include gates that modulate the passage of molecules in and out of the enzyme's active site. The enzyme PA1024 from Pseudomonas aeruginosa PA01 is a recently discovered flavin-dependent NADH:quinone oxidoreductase (NQO, EC 1.6.5.9). Q80 in loop 3 (residues 75-86) of NQO is ∼15 Å away from the flavin and creates a gate that seals the active site through a hydrogen bond with Y261 upon NADH binding. In this study, we mutated Q80 to glycine, leucine, or glutamate to investigate the mechanistic significance of distal residue Q80 in NADH binding in the active site of NQO. The UV-visible absorption spectrum reveals that the mutation of Q80 minimally affects the protein microenvironment surrounding the flavin. The anaerobic reductive half-reaction of the NQO-mutants yields a ≥25-fold increase in the K d value for NADH compared to the WT enzyme. However, we determined that the k red value was similar in the Q80G, Q80L, and wildtype enzymes and only ∼25% smaller in the Q80E enzyme. Steady-state kinetics with NQO-mutants and NQO-WT at varying concentrations of NADH and 1,4-benzoquinone establish a ≤5-fold decrease in the k cat /K NADH value. Moreover, there is no significant difference in the k cat /K BQ (∼1 × 10 6  M -1 s -1 ) and k cat (∼24 s -1 ) values in NQO-mutants and NQO-WT. These results are consistent with the distal residue Q80 being mechanistically essential for NADH binding to NQO with minimal effect on the quinone binding to the enzyme and hydride transfer from NADH to flavin., Competing Interests: Conflict of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher k cat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas K m values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity ( k cat /K m) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. Significance of the study Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle. [ABSTRACT FROM AUTHOR]

Flavocytochrome b2 (fcb2) (EC 1.1.2.3) is a 4 × 58 kD homotetrameric FMN and heme containing enzyme localized in the intermembrane space of yeast mitochondria. During the catalysis, FMN oxidizes L-lactate to pyruvate. Subsequently, the reduced FMN transfers electrons to the heme cofactor, which subsequently reduces cytochrome c. The kinetic properties and thermostability of the enzyme depend on its source, and these factors may be important in the construction of biosensors for L-lactate based on fcb2. In this work, we have investigated the kinetic properties of fcb2 from the thermotolerant methanotrophic strain Hansenula polymorpha. Like in our previous studies of Saccharomyces cerevisiae fcb2 [1], H. polymorpha fcb2 exhibited biphasic Lineweaver-Burk plots at fixed L-lactate concentrations using varied ferricyanide concentrations. It reflects the acceptance of electrons from both the reduced FMN and heme after the slower intramolecular FMN-to-heme electron transfer. At the infinite L-lactate concentration, the maximal rate of ferricyanide reduction is close to 120 s-1. The maximal rate of cytochrome c reduction is close to the second (slower) phase of ferricyanide reduction, because cytochrome c accepts electrons only from heme. The reaction product pyruvate inhibits H. polymorpha fcb2 in a mixed way at the millimolar concentration, and decreases the maximal rates of both fast and slow phases of ferricyanide reduction. [ABSTRACT FROM AUTHOR]

Changes of Leu109 and Arg448 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have as yet not been associated with altered fitness. However, in a recent study, we described that the simultaneous substitution of L109 and R448 by methionine leads to an error-producing polymerase phenotype that is not observed for the isolated substitutions. The double mutant increased the error rate of DNA-dependent DNA synthesis 3.1-fold as compared to the wildtype enzyme and showed a mutational spectrum with a fraction of 28% frameshift mutations and 48% transitions. We show here that weaker binding of DNA:DNA primer-templates as indicated by an increased dissociation rate constant ( k off) could account for the higher frameshift error rate. Furthermore, we were able to explain the prevalence of transition mutations with the finding that HIV-1 RT variant L109M/R448M preferred misincorporation of C opposite A and elongation of C:A mismatches. [ABSTRACT FROM AUTHOR]