Steady-state Kinetic Analysis Research Articles

Bm-iAANAT3: Expression and characterization of a novel arylalkylamine N-acyltransferase from Bombyx mori

The arylalkylamine N-acyltransferases (AANATs) are enzymes that catalyze the acyl-CoA-dependent formation of N-acylarylalkylamides: acyl-CoA + arylalkylamine → N-acylarylalkylamides + CoA-SH. Herein, we describe our study of a previously uncharacterized AANAT from Bombyx mori: Bm-iAANAT3. Bm-iAANAT3 catalyzes the direct formation of N-acylarylalkylamides and accepts a broad range of short-chain acyl-CoA thioesters and amines as substrates. Acyl-CoA thioesters possessing an acyl chain length >10 carbon atoms are not substrates for Bm-iAANAT3. We report that Bm-iAANAT3 is a “versatile generalist”, most likely, functioning in amine acetylation – a reaction in amine inactivation/excretion, cuticle sclerotization, and melanism. We propose a kinetic and chemical mechanism for Bm-iAANAT3 that is consistent with our steady-state kinetic analysis, dead-end inhibition studies, determination of the pH-rate profiles, and site-directed mutagenesis of a catalytically important amino acid in Bm-iAANAT3. These mechanistic studies of Bm-iAANAT3 will foster the development of novel compounds targeted against this enzyme and other insect AANATs for the control of insect pests.

Kinetic characterization of acetone monooxygenase from Gordonia sp. strain TY-5

Acetone monooxygenase (ACMO) is a unique member of the Baeyer–Villiger monooxygenase (BVMO) family based on its ability to act on small ketones, such as acetone. Herein, we performed a kinetic analysis of ACMO from the propane-utilizing bacterium Gordonia sp. strain TY-5 to assess its preference for smaller ketone substrates. Steady state kinetic analysis of ACMO with a range of linear (C3–C7) and cyclic ketones (C4–C6) reveals that the enzyme elicits the highest catalytic efficiency towards butanone and cyclobutanone. Stopped-flow and inhibition studies further revealed that ACMO has a relatively weak binding affinity for the coenzyme with a dissociation constant of 120 μM. We show through mutagenesis that sequence variation in the residue that coordinates to the 2′-phosphate of NADP(H) partially accounts for the weaker binding affinity observed. As for shown for related BVMOs, NADP+ stabilizes the C4a-peroxyflavin intermediate in ACMO; however, the rate of its formation is considerably slower in ACMO. The observed rate constant for NADPH-dependent flavin reduction was 60 s−1 at 25 °C, and experiments performed with 4(R)-[4-2H]NADPH confirm that the C4-pro-R-hydride from the nicotinamide ring is transferred to the FAD. The latter experimental result suggests that the nicotinamide ring rotates within the active site to carry out its two functional roles: reduction of the FAD cofactor and stabilization of the C4a-peroxyflavin adduct.

Highly efficient oxidation of synthetic and natural lignin-related compounds by Physisporinus vitreus versatile peroxidase

Versatile peroxidases (VPs) have outstanding oxidative capacity for a wide range of substrates and potential applications in industrial sectors and more. In this study, the VP1 from Physisporinus vitreus PF18 was obtained and overexpressed as inclusion body in Escherichia coli, then refolded successfully in vitro. Steady-state kinetic analysis and dye decolorization indicated that Mn(II) could promote the oxidation of low redox potential substrates and increase decolorization efficiency up to 70% by recombinant VP1 (rVP1). Results of pyrolysis-gas chromatography mass spectroscopy showed that most derivatives of G-type lignin increased slightly by rVP1 treatment, compared with the control. Surprisingly, the ratio of guaiacyl-type to syringyl-type derivatives (G/S) after rVP1 treatment was 5.4 times higher than that of the control. The polymerization of alkali lignin may be attributed to the transformation of S-type into G-type lignin by demethoxylation. Thus, our study provides a high-efficient candidate for biodegradation and biocatalysis.

Rational design of a stable peroxidase mimic for colorimetric detection of H2O2 and glucose: A synergistic CeO2/Zeolite Y nanocomposite

Owing to the high costs and instability of natural enzymes, the development of enzyme mimics based on inorganic nanomaterials has attracted a wide concern in recent years. In this work, a stable nanocomposite composed of highly dispersed CeO2 nanoparticles decorated on zeolite Y as support (CeO2/Y) was synthesized by a facile wet impregnation method, and the CeO2/Y nanocomposite was firstly proposed as an efficient peroxidase-mimicking nanozyme for accurate detection of H2O2 and glucose. The best catalyst was the nanocomposite with CeO2 to zeolite Y mass ratio of 0.20 (denoted as 20CeO2/Y), showing a better affinity and higher catalytic constant to the substrate of H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB) than horseradish peroxidase (HRP) enzymes by the steady-state kinetic analysis. The enzyme-mimicking catalyst could be used over a wider range of pH and temperature for a long-time reuse in TMB oxidation. A facile colorimetric assay was set up for the accurate detection of H2O2 and glucose, with the detection limits of 0.323 μM and 35.4 μM, respectively. The CeO2/Y-based peroxidase mimic was used to precisely detect the glucose concentration in real blood serum samples, exhibiting a great potential to be constructed as a reliable biosensor for glucose detection in some complex systems. The superior peroxidase-mimicking performance of CeO2/Y nanocomposite is attributed to the synergistic effects of outstanding activity of highly dispersed CeO2 nanoparticles (5–10 nm) and adsorption properties of zeolite Y with large surface area (517 m2·g−1) and pore volume (0.329 cm3·g−1).

Hydrogen-Bonding Interactions at the DNA Terminus Promote Extension from Methylguanine Lesions by Human Extender DNA Polymerase ζ.

Chemically induced DNA lesions can become DNA replication substrates that are bypassed by low-fidelity DNA polymerases. Following nucleotide misinsertion opposite a DNA lesion, the extension step can contribute to preserving such errors and lead to genomic instability and cancer. DNA polymerase ζ, a B-family polymerase, is proficient as an extender polymerase that catalyzes elongation; however, the chemical factors that impact its DNA replication are not understood. This study addresses the question of how DNA polymerase ζ achieves extension by examining the ability of recombinant human DNA polymerase ζ to extend from a series of methylated guanine lesions. The influence of H-bonding was examined by placing structurally altered nucleoside analogues and canonical bases opposite G, O6-MeG, N1-MeG, and N2-MeG. We determined that terminal base pairs with the highest proclivity for H-bonding were most efficiently extended in both primer extension assays and steady-state kinetic analysis. In contrast, when no H-bonding was possible at the DNA terminus, the least efficient steady-state kinetics were observed. To evaluate H-bonding protein minor groove interactions that may underlie this phenomenon, we performed computational modeling with Escherichia coli DNA polymerase II, a homologue for DNA polymerase ζ. The modeling data together with the primer extension assays demonstrate the importance of having a carbonyl group on the primer strand that can interact with a lysine residue found to be conserved in many B-family polymerases, including human Pol ζ. These data provide a model whereby interbase H-bonding interactions at the DNA terminus promote lesion bypass and extension by human DNA polymerase ζ.

Kinetic Understanding of N2 Reduction versus H2 Evolution at the E4(4H) Janus State in the Three Nitrogenases.

The enzyme nitrogenase catalyzes the reduction of N2 to ammonia but also that of protons to H2. These reactions compete at the mechanistically central 'Janus' intermediate, denoted E4(4H), which has accumulated 4e-/4H+ as two bridging Fe-H-Fe hydrides on the active-site cofactor. This state can lose e-/H+ by hydride protonolysis (HP) or become activated by reductive elimination ( re) of the two hydrides and bind N2 with H2 loss, yielding an E4(2N2H) state that goes on to generate two NH3 molecules. Thus, E4(4H) represents the key branch point for these competing reactions. Here, we present a steady-state kinetic analysis that precisely describes this competition. The analysis demonstrates that steady-state, high-electron flux turnover overwhelmingly populates the E4 states at the expense of less reduced states, quenching HP at those states. The ratio of rate constants for E4(4H) hydride protonolysis ( kHP) versus reductive elimination ( kre) provides a sensitive measure of competition between these two processes and thus is a central parameter of nitrogenase catalysis. Analysis of measurements with the three nitrogenase variants (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase) reveals that at a fixed N2 pressure their tendency to productively react with N2 to produce two NH3 molecules and an accompanying H2, rather than diverting electrons to the side reaction, HP production of H2, decreases with their ratio of rate constants, k re/ kHP: Mo-nitrogenase, 5.1 atm-1; V-nitrogenase, 2 atm-1; and Fe-nitrogenase, 0.77 atm-1 (namely, in a 1:0.39:0.15 ratio). Moreover, the lower catalytic effectiveness of the alternative nitrogenases, with more H2 production side reaction, is not caused by a higher kHP but by a significantly lower k re.

Kinetic analysis of cellulose synthase of Gluconacetobacter hansenii in whole cells and in purified form

The Gram-negative bacterium, Gluconacetobacter hansenii, has been long studied and is a model for cellulose synthesis. It produces cellulose, using the enzyme AcsA-AcsB, of exceptionally high crystallinity in comparison to the cellulose of higher plants. We determined the rate of cellulose synthesis in whole cells measured as moles of glucose incorporated into cellulose per second per mole of enzyme. This was determined by quantifying the rate of cellulose synthesis (over a short time span, such that the enzyme concentration is not changing due to cell growth) and the amount of enzyme in the whole cell by quantitative western blotting. We found that the whole cell rate of 24 s−1 is much faster than the kcat, measured from steady-state kinetic analysis, of 1.7 s−1. Our whole cell rates are consistent with previous studies using microscopy. We postulate that the rationale for this difference is the presence of an alternative in vivo priming mechanism. This in turn can increase the rate of initiation, which we previously postulated to be the rate-limiting step in catalysis.

Characteristics of refold acid urease immobilized covalently by graphene oxide-chitosan composite beads

Bifunctional acid urease can not only remove urea from rice wine, but also effectively remove ethyl carbamate (EC), thereby reducing harmful substances in rice wine. Acid urease from Providencia rettgeri JN-B815 was expressed in Escherichia coli BL21(DE3) as an inclusion body. Subsequently, acid urease was refolded gradually using dilution-ultrafiltration and specific activity of 12.3Umg-1 was obtained. The acid urease thus obtained was immobilized on graphene oxide/chitosan beads. The recovery of immobilized urease was 77% for the graphene oxide/chitosan composite when coupled for 8h with 5% glutaraldehyde. Steady-state kinetic analysis showed that the optimum temperature for urease activity was 37°C. Time-dependent thermal inactivation studies showed that the stability of the immobilized urease improved at 37°C and the t1/2 (half-life) of decay in urease activity was 12h, whereas it was 43 days for soluble and chitosan/graphene oxide immobilized urease at 4°C. A significant change in Km occurred during the immobilization; the Km for urea and EC in the free state were 9.14mM and 386mM, respectively, whereas they were 5.28mM and 180mM, respectively, in the immobilized state. The values of Vmax for immobilized EC and urea were 1.08μmolmin-1 and 1.16μmolmin-1, respectively, whereas they were 0.782μmolmin-1 and 0.931μmolmin-1, respectively, for soluble EC and urea. Furthermore, 90% of the original activity of graphene oxide/chitosan-urease beads were retained after the beads were used 10 times, indicating excellent reusability.

Kinetic Solvent Viscosity Effects as Probes for Studying the Mechanisms of Enzyme Action.

The study of enzyme reaction mechanisms is fundamentally important to our understanding of biochemistry, cellular metabolism, and drug development. This Perspective focuses on the use of kinetic solvent viscosity effects (KSVEs) to study enzyme reactions. This technique is easily implemented and uses steady-state kinetic analyses to probe whether substrate binding is diffusion-controlled and whether product release is the rate-limiting step in the catalytic cycle. In addition, KSVEs can identify isomerization steps that are important for catalysis. The use of KSVEs in combination with other techniques, such as kinetic isotope effects, pH effects, and site-directed mutagenesis, can provide a detailed view of the mechanism of enzyme action. We present the basic theory, important experimental considerations, and potential outcomes and briefly discuss some examples from the literature. The derivation of the equations that are important for data analysis is also presented.

Reaction Mechanism of Prephenate Dehydrogenase from the Alternative Tyrosine Biosynthesis Pathway in Plants.

Unlike metazoans, plants, bacteria, and fungi retain the enzymatic machinery necessary to synthesize the three aromatic amino acids l-phenylalanine, l-tyrosine, and l-tryptophan de novo. In legumes, such as soybean, alfalfa, and common bean, prephenate dehydrogenase (PDH) catalyzes the tyrosine-insensitive biosynthesis of 4-hydroxyphenylpyruvate, a precursor to tyrosine. The three-dimensional structure of soybean PDH1 was recently solved in complex with the NADP+ cofactor. This structure allowed for the identification of both the cofactor- and ligand-binding sites. Here, we present steady-state kinetic analysis of twenty site-directed active-site mutants of soybean (Glycine max) PDH compared to wild-type. Molecular docking of the substrate, prephenate, into the active site of the enzyme revealed its potential interactions with the active site residues and made a case for the importance of each residue in substrate recognition and/or catalysis, most likely through transition state stabilization. Overall, these results suggested that the active site of the enzyme is highly sensitive to any changes, as even subtle alterations substantially reduced the catalytic efficiency of the enzyme.

Structural and functional delineation of aerobactin biosynthesis in hypervirulent Klebsiella pneumoniae

Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of Klebsiella pneumoniae (hvKP). In contrast to classical opportunistic strains of K. pneumoniae, hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent "super bug." Despite hvKP harboring four distinct siderophore operons, knocking out production of only aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. In vivo heterologous production and in vitro reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.

Staphylopine, pseudopaline, and yersinopine dehydrogenases: A structural and kinetic analysis of a new functional class of opine dehydrogenase

Opine dehydrogenases (ODHs) from the bacterial pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Yersinia pestis perform the final enzymatic step in the biosynthesis of a new class of opine metallophores, which includes staphylopine, pseudopaline, and yersinopine, respectively. Growing evidence indicates an important role for this pathway in metal acquisition and virulence, including in lung and burn-wound infections (P. aeruginosa) and in blood and heart infections (S. aureus). Here, we present kinetic and structural characterizations of these three opine dehydrogenases. A steady-state kinetic analysis revealed that the three enzymes differ in α-keto acid and NAD(P)H substrate specificity and nicotianamine-like substrate stereoselectivity. The structural basis for these differences was determined from five ODH X-ray crystal structures, ranging in resolution from 1.9 to 2.5 Å, with or without NADP+ bound. Variation in hydrogen bonding with NADPH suggested an explanation for the differential recognition of this substrate by these three enzymes. Our analysis further revealed candidate residues in the active sites required for binding of the α-keto acid and nicotianamine-like substrates and for catalysis. This work reports the first structural kinetic analyses of enzymes involved in opine metallophore biosynthesis in three important bacterial pathogens of humans.

Discovery of a Kojibiose Phosphorylase in Escherichia coli K-12.

The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes ( ycjM-W and ompG) of unknown function in Escherichia coli K-12 were determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and β-d-glucose-1-phosphate ( kcat = 1.1 s-1, Km = 1.05 mM, and kcat/ Km = 1.12 × 103 M-1 s-1). Additionally, it was also shown that in the presence of β-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a β-phosphoglucomutase that catalyzes the isomerization of β-d-glucose-1-phosphate ( kcat = 21 s-1, Km = 18 μM, and kcat/ Km = 1.1 × 106 M-1 s-1) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with β-d-allose-1-phosphate, β-d-mannose-1-phosphate, and β-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-d-glucose-d-glycerate with a kcat = 2.1 s-1, Km = 69 μM, and kcat/ Km = 3.1 × 104 M-1 s-1.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Allosteric p97 inhibitors to overcome ATP‐competitive inhibitors resistance in anticancer therapy

Resistance mutations occurring on desired targets inside cancer cells, leading to loss of treatment effectiveness is a major challenge of targeted therapy; therefore, developing related small molecules with different mechanisms of action at different binding sites could be an effective strategy to overcome the resistant mutations. The ATPase enzyme, p97, is a promising target for cancer therapy because it is vital to cancer cell growth and survival. Development of resistance to existing ATP‐competitive p97 inhibitors such as CB‐5083 inspired identification of p97 inhibitors with different mechanisms of action. One approach to overcome CB‐5083 resistance is to use different classes of p97 inhibitors and therefore, we determined the potency of four ATP competitive p97 inhibitors (DBeQ, ML240, ML241, and CB‐5083) and two allosteric p97 inhibitors (NMS‐873 and UPCDC‐30245) using recombinantly purified CB‐5083 resistant p97 mutants and CB‐5083 resistant HCT116 colon cancer cells. Our results demonstrated that two known CB‐5083 resistant mutants (N660K and T688A p97) were consistently resistant to ATP‐competitive p97 inhibitors whereas allosteric p97 inhibitors NMS‐873 and UPCDC‐30245 were still active toward these mutants. The binding affinity of CB‐5083 to p97 mutants N660K and T688A is lower than to WT p97, which may be the mechanism of CB‐5083 drug resistance. To evaluate the effect of a single site mutation at N660 or T688 on p97 itself, we conducted steady state kinetic analysis. The results suggested that these mutations change the conformation of the p97 ATPase. N660K mutation decreased the Km of ATP to p97 whereas T688A decreased kcat of ATP hydrolysis. While CB‐5083, NMS‐873 and UPCDC‐30245 all effectively inhibited parent HCT116 cancer cell proliferation, allosteric p97 inhibitors (NMS‐873 and UPCDC‐30245) exhibited similar potency in inhibiting CB‐5083 resistant HCT116 cancer cell proliferation, which was more than 50‐fold resistance to CB‐5083.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural basis for proficient nucleotide incorporation across a major cisplatin‐DNA lesion by human DNA polymerase kappa (polκ)

Cisplatin (cis‐diamminedichloroplatinum) is a widely used chemotherapeutic compound. It reacts with the N7 atoms of adjacent guanines in DNA to form the Pt‐1,2‐d(GpG) intrastrand cross‐link (Pt‐GG) as a major product. This bulky cisplatin adduct blocks DNA replication by replicative polymerases, thereby promoting apoptosis of rapidly dividing cancer cells. However, cancer cells have developed resistance to cisplatin, partly because of the bypass of cisplatin adducts by specialized DNA polymerases (mostly belonging to the Y‐family) through translesion synthesis (TLS). The human Y‐family DNA polymerase eta (polη) is very efficient in replicating opposite the Pt‐GG lesions, but not in extending after this adduct. Recently, it was shown that human Y‐family DNA polymerase kappa (polκ) is indispensable for DNA repair synthesis in dorsal root ganglion neurons exposed to cisplatin. Polκ gene knockdown experiments in human cells have also indicated the involvement of polκ in TLS of the Pt‐GG lesion. In this study, we investigated the bypass potential and nucleotide incorporation preference of polκ opposite DNA containing the Pt‐GG adduct and determined two crystal structures of polκ complexed with such DNA. The ternary complex structures represent two consecutive stages of lesion bypass: nucleotide insertion opposite the 5′G (Pt‐GG2) and primer extension immediately after the lesion (Pt‐GG3). Our in vitro DNA replication assays using polκ and DNA containing the Pt‐GG lesion indicate that polκ inserts the correct dCTP opposite the 5′G of Pt‐GG (Pt‐GG2), when a nucleotide is already inserted opposite the 3′G (Pt‐GG1). Polκ also inserts the correct dATP opposite the T base immediately after the Pt‐GG (Pt‐GG3). However, polκ is not able to insert a nucleotide opposite the 3′G of Pt‐GG (Pt‐GG1). On the other hand, polη is very efficient in replicating the Pt‐GG lesion at two insertion stages (Pt‐GG1 and Pt‐GG2), but the enzyme is blocked at the extension stage (Pt‐GG3). In terms of fidelity, we found that polκ has the fewest mis‐insertions compared to two other human Y‐family polymerases polη and polι. Our steady‐state kinetic analysis showed that polκ incorporates dCTP and dATP opposite the 5′G of Pt‐GG (Pt‐GG2) and T template 5′ to the lesion (Pt‐GG3), respectively, with very similar relative efficiencies of 0.15–0.20, which are only ~5 fold lower than for normal DNA replication (GG2 and GG3). Our biochemical data indicate that polκ is very proficient and accurate in inserting a nucleotide after the first G of the Pt‐GG lesion. The structures revealed that the accuracy is achieved by stably accommodating the bases containing the cisplatin adducts in the enzyme's active site for proper Watson‐Crick base pairing with the incoming nucleotide. Together, our biochemical and structural data provide the molecular basis for the accurate and proficient incorporation by polκ of nucleotides opposite the cisplatin adduct and suggest an important role of polκ as a second polymerase (extender) in the efficient bypass of the Pt‐GG lesion in vivo. This work holds promise for polκ as a potential target for drug design, along with polη, which together could improve the efficacy of cisplatin treatment for cancer therapy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An Antarctic Extreme Halophile and Its Polyextremophilic Enzyme: Effects of Perchlorate Salts.

Effects of perchlorate salts prevalent on the surface of Mars are of significant interest to astrobiology from the perspective of potential life on the Red Planet. Halorubrum lacusprofundi, a cold-adapted halophilic Antarctic archaeon, was able to grow anaerobically on 0.04 M concentration of perchlorate. With increasing concentrations of perchlorate, growth was inhibited, with half-maximal growth rate in ca. 0.3 M NaClO4 and 0.1 M Mg(ClO4)2 under aerobic conditions. Magnesium ions were also inhibitory for growth, but at considerably higher concentrations, with half-maximal growth rate above 1 M. For a purified halophilic β-galactosidase enzyme of H. lacusprofundi expressed in Halobacterium sp. NRC-1, 50% inhibition of catalytic activity was observed at 0.88 M NaClO4 and 0.13 M Mg(ClO4)2. Magnesium ions were a more potent inhibitor of the enzyme than of cell growth. Steady-state kinetic analysis showed that Mg(ClO4)2 acts as a mixed inhibitor (KI = 0.04 M), with magnesium alone being a competitive inhibitor (KI = 0.3 M) and perchlorate alone acting as a very weak noncompetitive inhibitor (KI = 2 M). Based on the estimated concentrations of perchlorate salts on the surface of Mars, our results show that neither sodium nor magnesium perchlorates would significantly inhibit growth and enzyme activity of halophiles. This is the first study of perchlorate effects on a purified enzyme. Key Words: Halophilic archaea—Perchlorate—Enzyme inhibition—Magnesium. Astrobiology 18, 412–418.

Structural basis for substrate recognition and inhibition of prephenate aminotransferase from Arabidopsis.

Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (PAT) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition of PAT are currently unknown. Here we present the X-ray crystal structures of the wild-type and various mutants of PAT from Arabidopsis thaliana (AtPAT). Steady-state kinetic and ligand-binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution of PAT activity among class Ib AAT enzymes. Structures of AtPAT mutants co-crystallized with either α-ketoglutarate or pyridoxamine 5'-phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor of PAT from A. thaliana and Antirrhinum majus plants as well as the bacterium Chlorobium tepidum, uncovering a potential new effector of PAT.

Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not an Oxygenase.

The PLP-dependent l-arginine hydroxylase/deaminase MppP from Streptomyces wadayamensis (SwMppP) is involved in the biosynthesis of l-enduracididine, a nonproteinogenic amino acid found in several nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of l-arginine to form a mixture of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady-state kinetics analysis in the presence and absence of catalase shows that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid produced, two molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either [18]O2 or H2[18]O and analyzing the products by ESI-MS. Incorporation of [18]O was only observed when the reaction was performed in H2[18]O. Crystal structures of SwMppP with l-arginine, 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at resolutions of 2.2, 1.9. and 1.8 Å, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of N-terminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of l-arginine.

15(V/K) kinetic isotope effect and steady-state kinetic analysis for the transglutaminase 2 catalyzed deamidation and transamidation reactions

The Ca2+-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 NH3 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.