OptZyme: Computational Enzyme Redesign Using Transition State Analogues

OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., k_cat or k_cat/K_M) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that K_M correlates (R² = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, k_cat/K_M correlates (R² = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and k_cat correlates (R² = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved K_M, k_cat, and k_cat/K_M for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving K_M, k_cat, or k_cat/K_M. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S.     

Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K_M^DXP = 104 µM, K_M^NADPH = 13 µM, k_cat^DXP = 2 s⁻¹, k_cat^NADPH = 1.3 s⁻¹), an IC₅₀ for fosmidomycin of 247 nM, and a K_i for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg⁺² as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.     

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

Atrazine chlorohydrolase, TrzN (triazine hydrolase or atrazine chlorohydrolase 2), initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring. The present study describes crystal structures and reactivity of wild-type and active site mutant TrzN enzymes. The homodimer native enzyme structure, solved to 1.40 Å resolution, is a (βα)₈ barrel, characteristic of members of the amidohydrolase superfamily. TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members. The threonine side chain oxygen atom is 3.3 Å from the zinc atom and 2.6 Å from the oxygen atom of zinc-coordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in k_cat/K_m, largely due to k_cat, whereas the T325D and T325E mutants had immeasurable activity. The structure and kinetics of TrzN are reminiscent of carbonic anhydrase, which uses a threonine to assist in positioning water for reaction with carbon dioxide. An isosteric substitution in the active site glutamate, E241Q, showed a large diminution in activity with ametryn, no detectable activity with atratone, and a 10-fold decrease with atrazine, when compared with wild-type TrzN. Activity with the E241Q mutant was nearly constant from pH 6.0 to 10.0, consistent with the loss of a proton-donating group. Structures for TrzN-E241Q were solved with bound ametryn and atratone to 1.93 and 1.64 Å resolution, respectively. Both structure and kinetic determinations suggest that the Glu²⁴¹ side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.     

Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k_cat/K_m) compared to wild-type CA130. Their K_m values were decreased by ca. 50%, and the k_cat values increased to 14.4 and 16.9 s⁻¹, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.     

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k_cat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s^–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k_cat for ATP hydrolysis by 7-fold, to 0.32 s^–1, while not significantly altering the apparent K_m. The specificity constant for the interaction between ATP and topo IIβ (k_cat/K_mapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.     

Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation

This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9₂₅-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9₂₅-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5′-product terminating in a 2′,3′-phosphodiester and a 3′-product terminating in a 5′-OH. Under single turnover conditions at 24°C, 9₂₅-11t displays a maximum first-order rate constant, k_cat, of 0.037 min⁻¹ and a catalytic efficiency, k_cat/K_m, of 5.3 × 10⁵ M⁻¹ min⁻¹. The measured value of k_cat under catalyst excess conditions agrees with the value of k_cat observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9₂₅-11t was gauged in terms of k_cat values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k_cat values by a factor of 4 to 250, indicating that 9₂₅-11t displays significant sequence specificity despite the lack of an apparent Watson–Crick base-pairing scheme for recognition.     

Characterization of an Acidic Chitinase from Seeds of Black Soybean (Glycine max (L) Merr Tainan No. 3)

Using 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside (4-MU-GlcNAc₃) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAc_n, n = 3-5), and pNp-GlcNAc₄ was the most degradable substrate. Using pNp-GlcNAc₄ as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters K _m and k_cat were 245 µM and 10.31 min⁻¹, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides.     

Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD⁺ salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (k_cat/K_m = 43.5 mM⁻¹s⁻¹) than pyrazinamidase (k_cat/K_m = 3.2 mM⁻¹s⁻¹). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.     

Insights into β-Lactamases from Burkholderia Species,Two Phylogenetically Related yet Distinct Resistance Determinants

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with k_cat values ranging from 0.38 ± 0.04 to 460 ± 46 s⁻¹ and possesses high k_cat/k_inact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest k_cat value for cefotaxime of 9.0 ± 0.9 s⁻¹. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k_cat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.     

Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site‐directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in k _cat relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in k _cat/K _M across the series. Only modest decreases in k _cat/K _M are observed for the E85Q variants with little effect on k _cat. Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst.     

Engineering of a Type III Rubisco from a Hyperthermophilic Archaeon in Order To Enhance Catalytic Performance in Mesophilic Host Cells

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbc_Tk). It has previously been shown that Rbc_Tk is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris Δ3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of Rbc_Tk at 25°C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and α-helix 6 regions among Rbc_Tk and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of Rbc_Tk is one residue longer than those found in the spinach and G. partita enzymes, and replacing Rbc_Tk loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris Δ3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type Rbc_Tk. SP6 corresponds to a complete substitution of the original α-helix 6 of Rbc_Tk with that of the spinach enzyme. Compared to wild-type Rbc_Tk, the purified SP6 mutant protein exhibited a 30% increase in turnover number (k_cat) of the carboxylase activity and a 17% increase in the k_cat/K_m value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in Rbc_Tk and also led to the identification of specific residue changes that resulted in an increase in the turnover number of Rbc_Tk at ambient temperatures.     

Slow Axonemal Dynein e Facilitates the Motility of Faster Dynein c

We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (k_cat = 0.27 s^–1 and k_cat,MT = 1.09 s^–1, respectively) were lower than those of dynein c (k_cat = 1.75 s^–1 and k_cat,MT = 2.03 s^–1, respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (V_ap = 1.2 ± 0.1 μm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (V_max = 15.8 ± 1.5 μm/s) and highly sensitive to decreases in the surface density (V_min = 2.2 ± 0.7 μm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c’s power stroke.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Structural and Kinetic Characterization of 4-Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolase,a Protocatechuate Degradation Enzyme Evolutionarily Convergent with the HpaI and DmpG Pyruvate Aldolases

4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate α-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of k_cat/K_m and k_cat for the enzyme is consistent with a single deprotonation with pK_a values of 8.0 ± 0.1 and 7.0 ± 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 Å x-ray structure shows a four-layered α-β-β-α sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199′. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.     

Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO₂ ^– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are k _app1 = 9.6±0.2 M^–1 s^–1 and k _app2 = 1.2±0.1 M^–1 s^–1 (at pH 7.4 and 20°C). The k _app1 and k _app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h _app = 3.8×10⁴ M^–1 s^–1 and h ₀ = 2.8×10^–1 s^–1 (at pH 7.4 and 20°C). The pH-dependence of h _on and h ₀ values reflects the acid-base equilibrium of peroxynitrite (pK _a = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.     

Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism [Biochimica et Biophysica Acta 1597 (2002) 157-163]

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (α9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the α9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k_cat and k_cat/K_m for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the α9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Δ⁵-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k_cat/K_m. The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.     

Effects of Familial Mutations on the Monomer Structure of Aβ42

Amyloid beta (Aβ) peptide plays an important role in Alzheimer’s disease. A number of mutations in the Aβ sequence lead to familial Alzheimer’s disease, congophilic amyloid angiopathy, or hereditary cerebral hemorrhage with amyloid. Using molecular dynamics simulations of ∼200 μs for each system, we characterize and contrast the consequences of four pathogenic mutations (Italian, Dutch, Arctic, and Iowa) for the structural ensemble of the Aβ monomer. The four familial mutations are found to have distinct consequences for the monomer structure.Amyloid beta (Aβ) peptides have long been thought to play a central role in Alzheimer’s disease (AD). Usually 40 or 42 residues in length, Aβ peptides are proteolytic products of the Aβ precursor protein and they aggregate to form the fibrillar plaques in AD patients’ brains. Besides fibrillar plaques, Aβ oligomers are also neurotoxic. The significance and nature of Aβ oligomerization has recently become a focus of intensive research studies and debates (1,2). Notably, numerous pathogenic mutations have been identified in the Aβ precursor protein sequence and in the enzymes involved in Aβ processing (3). These mutations generally lead to early onset of AD or cerebral amyloid angiopathy. Understanding how the pathogenic mutations alter Aβ oligomerization/aggregation is essential to our understanding of the disease mechanism.Four of these pathogenic mutations (Italian E22K, Dutch E22Q, Arctic E22G, and Iowa D23N) cluster in the region of E22 and D23 in the Aβ sequence (distal from proteolytic cleavage sites) and they have higher neurotoxicity compared to wild-type (WT) Aβ (4). These mutations are thought to modify the physicochemistry of the peptide. For example, kinetic studies (4) show that the E22K and E22Q mutations lead to faster peptide aggregation, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (although the E22G mutation shows increased protofibril formation (5)). Recent solid-state NMR studies also suggest that rather than the in-register β-sheet conformation adopted by WT Aβ, the Iowa D23N mutant forms amyloid fibrils with antiparallel β-sheet structure (6).To understand how the mutations modify the peptide oligomerization/aggregation it is critical to characterize the starting point of the process, the monomers. Unfortunately, investigating the early phase of the oligomerization process experimentally is a challenging task due to the high aggregation propensity of Aβ and its intrinsic disorder. Therefore, a number of computational approaches have been adopted to investigate the consequences of mutations for the monomer structure (7–16). However, due to the high computational demands of explicit-solvent molecular dynamics (MD) simulations to simulate full-length Aβ peptides, most of these computational studies are either on Aβ fragments (to decrease the system size) using explicit-solvent simulations (8–12) or on full-length Aβ using implicit-solvent simulations (which are less computationally demanding and enable longer simulation times, but lack explicit water molecules in the simulations to fully describe water-peptide interactions) (13–15). In a very recent report, explicit-solvent simulations were used to study the effects of the E22Q mutation on full-length Aβ; however, rather limited data (<10 μs) were collected (16). Thus, characterizing full-length Aβ monomers remains quite a daunting task even with simulations.To characterize the effects of mutations on full-length Aβ monomer using explicit-solvent MD simulations, we employed distributed computing (17) to simulate the WT Aβ₄₂, Aβ₄₂-E22K, Aβ₄₂-E22Q, Aβ₄₂-E22G, and Aβ₄₂-D23N monomers. MD simulations of >200 μs were performed for each system and AMBER ff99sb (18) and the tip3p water model (19) were used for force field parameters. Peptide configurations in the MD trajectories were clustered with the root mean-square deviation metric to identify representative conformations (i.e., states) and transitions between these states were counted. Markov state model analysis was then performed where the master equations were solved and the equilibrium population of each state deduced (20). Details of the MD simulation procedures and Markov state model analysis can be found in the Supporting Material.Each of the five Aβ monomer systems exhibits great structural diversity and can only be characterized in an ensemble fashion (rather than described by a handful of representative configurations). This is in accord with the notion that full-length Aβ peptides are intrinsically disordered (21,22). Using the Dictionary of Secondary Structure of Proteins program (23) to assign secondary structure, it is clear that the five Aβ monomer systems are found overall not well structured, although small β-hairpins and α-helices are observed. In Fig. 1 we plot the residue-dependent extended β propensity and α-helix propensity, in the top and bottom panels, respectively, for each Aβ monomer system. Although we are reasonably confident of the convergence behavior of the α-helix propensity, we note that the convergence of the extended β-propensity might be more challenging and demand a much longer sampling time than the current aggregate simulation time of ∼200 μs (24).Open in a separate windowFigure 1Ensemble-averaged %population of β-strand (top) and α-helix (bottom) propensity for all five monomer systems. The sequence of the WT Aβ₄₂ is given on the x axis.We observe in Fig. 1 that all five Aβ monomer systems share a rather similar residue-dependent tendency to form an extended β-structure, although minor differences are present. On the other hand, these pathogenic mutations alter the α-helix propensity quite significantly. The E22K and E22Q mutations increase the α-helix propensity in the region of residues 20–23. All four mutations (E22K, E22Q, E22G, and D23N) decrease the α-helix propensity in the region of residues 33–36.Notably, we find that in all five systems only short stretches of α-helices are formed. That is, when a residue is involved in α-helix formation, it participates in forming mostly short helical segments (consisting of only four helical residues). To provide more insight into the changes of α-helix propensity due to the mutations, in Fig. S1 we plot the tendency of forming short α-helices along the sequence for all five systems. Each data point in Fig. S1 represents the propensity to form an α-helix of four residues in length, ending at the specific residue. For example, in the structural ensemble adopted by the WT peptide, ∼5.5% of the conformations have a short α-helix of size four, involving residues 15–18. We see from Fig. S1 that the E22K and E22Q mutations induce the formation of two short helices in residues 19–22 and 20–23. The higher α-helix propensity in this region for the E22K mutant compared to the WT was previously attributed to the elimination of the electrostatic repulsion between E22 and D23 in the WT by the mutation and the longer aliphatic chain of K22 in the mutant compared to E22 in the WT (9,22). This is consistent with the observation that the E22Q mutation also induces helix formation in this region (by eliminating the electrostatic repulsion between E22 and D23 in the WT) but to a lesser extent, possibly due to the shorter aliphatic chain of Q22 compared to K22.In the E22G mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, glycine is known to be a helix breaker (25), leading to diminished α-helix propensity in the region around residue G22 seen in Fig. S1.In the D23N mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, it does not induce (or rather even slightly decreases) helix formation around residue 23. This may be due to the short aliphatic chain of N23 but it is possible that the mutation induces some nonlocal effects on the peptide structure, disfavoring helix formation in this region.It is worth noting that all four mutations (E22K, E22Q, E22G, and D23N) virtually eliminate the α-helix propensity in the region of residues 33–36. This region is rather far away from the mutation sites in sequence but its α-helix propensity is nonetheless affected. The origin of such a nonlocal effect is less straightforward to explain and further analysis will aid untangling this behavior. Nonetheless, the diminished α-helix propensity in the region of residues 33–36 appears to be a consistent feature across all four mutants.The four mutations studied here (E22K, E22Q, E22G, and D23N) have been thought to modify the physicochemistry of the peptide and alter the oligomerization/aggregation process, leading to higher neurotoxicity. In predicting intrinsic aggregation propensities using peptide sequences, all four mutants are suggested to be more aggregation prone (26). On the other hand, kinetic studies show that only the E22K and E22Q mutants aggregate more quickly, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (4). Our simulation results suggest these pathogenic mutations have complicated effects on the monomer structure—all four mutations decrease helix propensity in residues 33–36, whereas only the E22K and E22Q mutations increase helix propensity in residues 20–23. It is interesting to note that α-helix propensity is generally thought to anticorrelate with aggregation propensity; however, recent studies have suggested an important role of α-helical intermediates in amyloid oligomerization (27–29). Our studies suggest that it would be of great value to investigate how the distinct patterns of α-helix propensity in these five systems may propagate to give rise to different oligomerization kinetics or even mechanisms. The pathogenic mutations studied here have complex effects on the oligomerization of the peptide. The characterization of the monomer structural ensembles reported here should aid understanding of such an important and complicated process.