Imparting As(III) Responsiveness to the Choline Response Transcriptional Regulator BetI.

The development of a low-cost and user-friendly sensor using microorganisms to monitor the presence of As(III) on earth has garnered significant attention. In conventional research on microbial As(III) sensors, the focus has been on transcription factor ArsR, which plays a role in As(III) metabolism. However, we recently discovered that LuxR, a quorum-sensing control factor in Vibrio fischeri that contains multiple cysteine residues, acted as an As(III) sensor despite having no role in As(III) metabolism. This finding suggested that any protein could be an As(III) sensor if cysteine residues were incorporated. In this study, we aimed to confer As(III) responsiveness to BetI, a transcriptional repressor of the TetR family involved in osmotic regulation of the choline response, unrelated to As(III) metabolism. Based on the BetI structure constructed using molecular dynamics calculations, we generated a series of mutants in which each of the three amino acids not critical for function was substituted with cysteine. Subsequent examination of their response to As(III) revealed that the cysteine-substituted mutant, incorporating all three substitutions, demonstrated As(III) responsiveness. This was evidenced by the fluorescence intensity of the downstream reporter superfolder green fluorescent protein expression regulated by the operator region. Intriguingly, the BetI cysteine mutant maintained its binding responsiveness to the natural ligand choline. We successfully engineered an OR logic gate capable of responding to two orthogonal ligands using a single protein.

Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases.

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

Spatially restricted substrate-binding site of cortisol-synthesizing CYP11B1 limits multiple hydroxylations and hinders aldosterone synthesis.

Human cytochromes P45011ß (CYP11B1) and P450aldo (CYP11B2) are monooxygenases that synthesize cortisol through steroid 11ß-hydroxylation and aldosterone through a three-step process comprising 11ß-hydroxylation and two 18-hydroxylations, respectively. CYP11B1 also catalyzes 18-monohydroxylation and 11ß,18-dihydroxylation. To study the molecular basis of such catalytic divergence of the two enzymes, we examined a CYP11B1 mutant (Mt-CYP11B1) with amino acid replacements on the distal surface by determining the catalytic activities and crystal structure in the metyrapone-bound form at 1.4-Å resolution. Mt-CY11B1 retained both 11ß-hydroxylase and 18-hydroxylase activities of the wild type (Wt-CYP11B1) but lacked 11ß,18-dihydroxylase activity. Comparisons of the crystal structure of Mt-CYP11B1 to those of Wt-CYP11B1 and CYP11B2 that were already reported show that the mutation reduced the innermost space putatively surrounding the C3 side of substrate 11-deoxycorticosterone (DOC) bound to Wt-CYP11B1, while the corresponding space in CYP11B2 is enlarged markedly and accessible to bulk water through a channel. Molecular dynamics simulations of their DOC-bound forms supported the above findings and revealed that the enlarged space of CYP11B2 had a hydrogen bonding network involving water molecules that position DOC. Thus, upon positioning 11ß-hydroxysteroid for 18-hydroxylation in their substrate-binding sites, steric hindrance could occur more strongly in Mt-CYP11B1 than in Wt-CYP11B1 but less in CYP11B2. Our investigation employing Mt-CYP11B1 sheds light on the divergence in structure and function between CYP11B1 and CYP11B2 and suggests that CYP11B1 with spatially-restricted substrate-binding site serves as 11ß-hydroxylase, while CYP11B2 with spatially-extended substrate-binding site successively processes additional 18-hydroxylations to produce aldosterone.

Structural and functional characterization of nylon hydrolases.

Biodegradation of synthetic polymers is recognized as a useful way to reduce their environmental load and pollution, loss of natural resources, extensive energy consumption, and generation of greenhouse gases. The potential use of enzymes responsible for the degradation of the targeted polymers is an effective approach which enables the conversion of the used polymers to original monomers and/or other useful compounds. In addition, the enzymes are expected to be applicable in industrial processes such as improving the surface structures of the polymers. Especially, conversion of the solid polymers to soluble oligomers/monomers is a key step for the biodegradation of the polymers. Regarding the hydrolysis of polyamides, three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (NylA), 6-aminohexanoate-dimer hydrolase (NylB), and 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC), are found in several bacterial strains. In this chapter, we describe our approach for the screening of microorganisms which degrade nylons and related compounds; preparation of substrates; assay of hydrolytic activity for soluble and insoluble substrates; and X-ray crystallographic and computational approaches for analysis of structure and catalytic mechanisms of the nylon-degrading enzymes.

First-Principles Study of the Reaction Mechanism of CHO + H on Graphene Surface.

Many organic molecules observed in the interstellar medium are considered to be formed on dust grains and populated into the gas phase. We analyzed the reaction of HCO + H on a graphene surface using ab initio molecular dynamics simulations as a case study of the formation and desorption of organic molecules on interstellar dust particles. During the reactions of chemisorbed CHO (chemisorbed at the C atom) with free H, CO was generated and efficiently desorbed from the surface. These results suggest that the reactions, of which the reactant forms a covalent bond with the surface while the product does not, cause efficient desorption of the product upon reaction. In such reactions a repulsive force between the product and the surface would be generated and accelerate translation of the product in a specific direction. In addition, it was also shown that the branching ratio of the reactions between radical species on the surface would be affected by the form of the adsorption on the surface, e.g., when a free H reacted with the CHO chemisorbed at the C atom, CH2O was not generated.

Molecular mechanisms of substrate specificities of uridinecytidine kinase.

A uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine (Urd) and cytidine (Cyd) and plays a significant role in the pyrimidine-nucleotide salvage pathway. Unlike ordinary ones, UCK from Thermus thermophilus HB8 (ttCK) loses catalytic activity on Urd due to lack of a substrate binding ability and possesses an unusual amino acid, i.e. tyrosine 93 (Tyr93) at the binding site, whereas histidine (His) is located in the other UCKs. Mutagenesis experiments revealed that a replacement of Tyr93 by His or glutamine (Gln) recovered catalytic activity on Urd. However, the detailed molecular mechanism of the substrate specificity has remained unclear. In the present study, we performed molecular dynamics simulations on the wild-type ttCK, two mutant ttCKs, and a human UCK bound to Cyd and three protonation forms of Urd to elucidate their substrate specificity. We found three residues, Tyr88, Tyr/His/Gln93 and Arg152 in ttCKs, are important for recognizing the substrates. Arg152 contributes to induce a closed form of the binding site to retain the substrate, and the N3 atom of Urd needed to be deprotonated. Although Tyr88 tightly bound Cyd, it did not sufficiently bind Urd because of lack of the hydrogen bonding. His/Gln93 complemented the interaction of Tyr88 and raised the affinity of ttCK to Urd. The crucial distinction between Tyr and His or Gln was a role in the hydrogen-bonding network. Therefore, the ability to form both hydrogen-bonding donor and accepter is required to bind both Urd and Cyd.

Mutations affecting the internal equilibrium of the reaction catalyzed by 6-aminohexanoate-dimer hydrolase.

UNLABELLED: The enzyme 6-aminohexanoate-dimer hydrolase catalyzes amide synthesis. The yield of this reverse reaction in 90% t-butyl alcohol was found to vary drastically when enzyme mutants with substitutions of several amino acids located at the entrance of the catalytic cleft were used. Movement of the loop region and the flip-flop of Tyr170 generate a local hydrophobic environment at the catalytic center of the enzyme. Here, we propose that the shift of the internal equilibrium between the enzyme-substrate complex and enzyme-product complex by the 'water-excluding effect' alters the rate of the forward and reverse reactions. Moreover, we suggest that the local hydrophobic environment potentially provides a reaction center suitable for efficient amide synthesis. DATABASE: PDB code 3VWL: Hyb-24DNY-S(187) PDB code 3VWM: Hyb-24DNY-A(187) PDB code 3VWN: Hyb-24DNY-G(187) PDB code 3A65: Hyb-24DN-A(112) /Ahx complex PDB code 3A66: Hyb-24DNY-A(112) /Ahx complex PDB code 3VWP: Hyb-24DNY-S(187) A(112) /Ahx complex PDB code 3VWQ: Hyb-24DNY-A(187) A(112) /Ahx complex PDB code 3VWR: Hyb-24DNY-G(187) A(112) /Ahx complex.

Unraveling the degradation of artificial amide bonds in nylon oligomer hydrolase: from induced-fit to acylation processes.

To elucidate how the nylon oligomer hydrolase (NylB) acquires its peculiar degradation activity towards non-biological amide bonds, we inspected the underlying enzymatic processes going from the induced-fit upon substrate binding to acylation. Specifically we investigated the mutational effects of two mutants, Y170F and D181G, indicated in former experiments as crucial systems because of their specific amino acid residues. Therefore, by adopting first-principles molecular dynamics complemented with metadynamics we provide a detailed insight into the underlying acylation mechanism. Our results show that while in the wild type (WT) the Tyr170 residue points the NH group towards the proton-acceptor site of an artificial amide bond, hence ready to react, in the Y170F this does not occur. The reason is ascribed to the absence of Tyr170 in the mutant, which is replaced by phenylalanine, which is unable to form hydrogen bond with the amide bond; thus, resulting in an increase in the activation barrier of more than 10 kcal mol(-1). Nonetheless, despite the lack of hydrogen bonding between the Y170F and the substrate, the highest free energy barrier for the induced-fit is similar to that of WT. This seems to suggest that in the induced-fit process, kinetics is little affected by the mutation. On the basis of additional structural homology analyses on the enzymes of the same family, we suggest that natural selection is responsible for the development of the peculiar hydrolytic activity of Arthrobacter sp. KI72.

A QM/MM study of the L-threonine formation reaction of threonine synthase: implications into the mechanism of the reaction specificity.

Threonine synthase catalyzes the most complex reaction among the pyridoxal-5'-phosphate (PLP)-dependent enzymes. The important step is the addition of a water molecule to the Cß-Cα double bond of the PLP-α-aminocrotonate aldimine intermediate. Transaldimination of this intermediate with Lys61 as a side reaction to form α-ketobutyrate competes with the normal addition reaction. We previously found that the phosphate ion released from the O-phospho-l-homoserine substrate plays a critical role in specifically promoting the normal reaction. In order to elucidate the detailed mechanism of this "product-assisted catalysis", we performed comparative QM/MM calculations with an exhaustive search for the lowest-energy-barrier reaction pathways starting from PLP-α-aminocrotonate aldimine intermediate. Satisfactory agreements with the experiment were obtained for the free energy profile and the UV/vis spectra when the PLP pyridine N1 was unprotonated and the phosphate ion was monoprotonated. Contrary to an earlier proposal, the base that abstracts a proton from the attacking water was the ε-amino group of Lys61 rather than the phosphate ion. Nevertheless, the phosphate ion is important for stabilizing the transition state of the normal transaldimination to form l-threonine by making a hydrogen bond with the hydroxy group of the l-threonine moiety. The absence of this interaction may account for the higher energy barrier of the side reaction, and explains the mechanism of the reaction specificity afforded by the phosphate ion product. Additionally, a new mechanism, in which a proton temporarily resides at the phenolate O3' of PLP, was proposed for the transaldimination process, a prerequisite step for the catalysis of all the PLP enzymes.

Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II.

The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3' OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.

Nylon-Oligomer Hydrolase Promoting Cleavage Reactions in Unnatural Amide Compounds.

The active site of 6-aminohexanoate-dimer hydrolase, a nylon-6 byproduct-degrading enzyme with a ß-lactamase fold, possesses a Ser112/Lys115/Tyr215 catalytic triad similar to the one of penicillin-recognizing family of serine-reactive hydrolases but includes a unique Tyr170 residue. By using a reactive quantum mechanics/molecular mechanics (QM/MM) approach, we work out its catalytic mechanism and related functional/structural specificities. At variance with other peptidases, we show that the involvement of Tyr170 in the enzyme-substrate interactions is responsible for a structural variation in the substrate-binding state. The acylation via a tetrahedral intermediate is the rate-limiting step, with a free-energy barrier of ∼21 kcal/mol, driven by the catalytic triad Ser112, Lys115, and Tyr215, acting as a nucleophile, general base, and general acid, respectively. The functional interaction of Tyr170 with this triad leads to an efficient disruption of the tetrahedral intermediate, promoting a conformational change of the substrate favorable for proton donation from the general acid.

An accurate density functional theory based estimation of pK(a) values of polar residues combined with experimental data: from amino acids to minimal proteins.

We report a scheme for estimating the acid dissociation constant (pK(a)) based on quantum-chemical calculations combined with a polarizable continuum model, where a parameter is determined for small reference molecules. We calculated the pK(a) values of variously sized molecules ranging from an amino acid to a protein consisting of 300 atoms. This scheme enabled us to derive a semiquantitative pK(a) value of specific chemical groups and discuss the influence of the surroundings on the pK(a) values. As applications, we have derived the pK(a) value of the side chain of an amino acid and almost reproduced the experimental value. By using our computing schemes, we showed the influence of hydrogen bonds on the pK(a) values in the case of tripeptides, which decreases the pK(a) value by 3.0 units for serine in comparison with those of the corresponding monopeptides. Finally, with some assumptions, we derived the pK(a) values of tyrosines and serines in chignolin and a tryptophan cage. We obtained quite different pK(a) values of adjacent serines in the tryptophan cage; the pK(a) value of the OH group of Ser13 exposed to bulk water is 14.69, whereas that of Ser14 not exposed to bulk water is 20.80 because of the internal hydrogen bonds.

Theoretical insight into stereoselective reaction mechanisms of 2,4-pentanediol-tethered ketene-olefin [2 + 2] cycloaddition.

We report ab initio molecular dynamics calculations based on density functional theory performed on an intramolecular [2 + 2] cycloaddition between ketene and olefin linked with a 2,4-pentanediol (PD) tether. We find that the encounter of the ketene and olefin moieties could be prearranged in the thermal equilibrated state before the cycloaddition. The reaction mechanism is found to be stepwise, similar to that of intermolecular ketene [2 + 2] cycloadditions with ordinary alkenes. A distinct feature of the reaction pathway for a major diastereoisomer is a differential activation free energy of about 1.5 kcal/mol, including 2.8 kcal/mol as the differential activation entropy, with a transition state consisting of a flexible nine-membered ring in the olefin-PD-ketene moiety. This theoretical study provides a reasonable explanation for the strict stereocontrollability of the PD-tethered ketene-olefin cycloaddition, irrespective of reaction types or conditions.

First-principles molecular dynamics study on the atomistic behavior of His503 in bovine cytochrome c oxidase.

We report first-principles molecular dynamics calculations based on density functional theory performed on the entrance part of the D-path pathway in bovine cytochrome c oxidase. Our models, which are extracted from the fully reduced and oxidized X-ray structures, include His503 as a protonatable site. We find that the protonated His503 with the deprotonated Asp91 [H503-N(δ1)H(+) and D91-C(γ)OO(γ)] are more energetically favorable than other protonation states, [H503-N(δ1) and D91-C(γ)OOH], with an energy difference of about -5kcal/mol in reduced case, while the [H503-N(δ1)H+ and D91-C(γ)OO(-)] state is energetically unstable, about +3kcal/mol higher in energy in the oxidized case. The local interaction of His503 with the surrounding polar residues is necessary and sufficient for determining the energetics. The redox-coupled rotation of His503 is found to change the energetics of the protonation states. We also find that this rotation is coupled with the proton transfer from His503 and Asp91, which leads to the transition between the two different protonation states. This study suggests that His503 is involved in the proton supply to the D-path as a proton acceptor and that the redox-controlled proton-transfer-coupled rotation of His503 is a key process for an effective proton supply to the D-path from water bulk. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Energy compensation mechanism for charge-separated protonation states in aspartate-histidine amino acid residue pairs.

The initial stage of proton propagation in the D-path channel of bovine cytochrome c oxidase, consisting of the approach of an H(+) to the entrance of this specific pathway, is inspected via first-principles calculations. Our model, extracted from the X-ray crystallographic structure, includes the amino acid residue pair aspartate (Asp91) and histidine (His503) as protonatable sites. Our calculations show that an additional proton, corresponding to the H(+) uptake by the enzyme from the inner bulk water, is transferred to either Asp91 or His503, leading to the formation of a neutral or a charge-separated protonation state. The relative stability between the two states amounts to a total energy difference of about 5 kcal/mol; this indicates that both Asp91 and His503 are involved in the proton supply to the D-path, playing the role of proton acceptors. The hydrogen-bond environment around Asp91 and His503 has an important influence on both the energetics and the electronic structure of the system; for instance, it compensates the Coulomb-energy cost in the charge-separated protonation state. An energy partitioning analysis shows that the compensatory effect is mainly due to local electrostatic interactions among the charged Asp91 and His503 side chains and the surrounding polar residues. The energy compensation mechanism we found in this work balances the energetics of Asp-His pairs, hence permitting an efficient and selective regulation of the protonatable amino acid residues, where several protonation states are accessible within energy differences of the order of a few H-bonds.

Significant change in electronic structures of heme upon reduction by strong Coulomb repulsion between Fe d electrons.

We report total-energy electronic-structure calculations based on the density functional theory performed on a low-spin heme. We have found that the high-lying occupied and low-lying unoccupied states having Fe d and/or porphyrin pi orbital character are significantly rearranged upon the reduction of the heme. An analysis of these states shows that the remarkable elevation of the Fe d levels takes place due to the strong Coulombic repulsion between accommodated d electrons. Due to a peculiarity of the heme, this elevation could be controlled by lower-lying empty porphyrin pi states, leading to electron transfer from Fe d orbitals to the porphyrin pi ones in order to reduce the Coulomb-energy cost. This self-limiting mechanism provides a natural explanation not only for the present calculated results, but also for general electron delocalization appearing under various physiological conditions, regardless of the types of the hemes.

Possible mechanism of proton transfer through peptide groups in the H-pathway of the bovine cytochrome c oxidase.

The peptide group connecting Tyr440 and Ser441 of the bovine cytochrome c oxidase is involved in a recently proposed proton-transfer path (H-path) where, at variance with other pathways (D- and K-paths), a usual hydrogen-bond network is interrupted, thus making this proton propagation rather unconventional. Our density-functional based molecular dynamics simulations show that, despite this anomaly and provided that a proton can reach a nearby water, a multistep proton-transfer pathway can become a viable pathway for such a reaction: a proton is initially transferred to the carbonyl oxygen of a keto form of the Tyr440-Ser441 peptide group [-CO-NH-], producing an imidic acid [-C(OH)-NH-] as a metastable state; the amide proton of the imidic acid is then transferred, spontaneously to the deprotonated carboxyl group of the Asp51 side chain, leading to the formation of an enol form [-C(OH)=N-] of the Tyr440-Ser441 peptide group. Then a subsequent enol-to-keto tautomerization occurs via a double proton-transfer path realized in the two adjacent Tyr440-Ser441 and Ser441-Asp442 peptide groups. An analysis of this multistep proton-transfer pathway shows that each elementary process occurs through the shortest distance, no permanent conformational changes are induced, thus preserving the X-ray crystal structure, and the reaction path is characterized by a reasonable activation barrier.

First-principles molecular dynamics study of proton transfer mechanism in bovine cytochrome c oxidase.

Density functional based first-principles molecular dynamics calculations, performed on a model system extracted from the bovine cytochrome c oxidase, have been performed in an attempt to inspect the proton transfer mechanism across a peptide group. Our model system includes the specific Tyr440-Ser441 peptide group involved in a novel proton transfer path and shows that the Y440-S441 enol peptide group [-C(OH) = N-], which is a structural isomer of a keto form [-CO-NH-], is the product of the deprotonation of an imidic acid [-C(OH)-NH-] occurring in the vicinity of the deprotonated aspartic acid residue. For the subsequent enol-to-keto tautomerization, a direct H(+) transfer path in the Y440-S441 peptide group has been identified, in which the transition state takes a distorted four-membered ring structure.

Enol-to-keto tautomerism of peptide groups.

Density functional based simulations, performed on polyglycine containing an enol peptide group [-C(OH)N-] which is a structural isomer of a keto form [-CONH-], show that in the enol-to-keto tautomeric reaction, the enol peptide group is less stable than the keto form, and that the enol-to-keto tautomerism is characterized by a cis/trans isomerization of the C-N peptide bond. The rate-limiting step in the cis/trans isomerization is a hydrogen migration from O to N atoms in the peptide group with a transition state consisting of a four-membered ring in the cis configuration. An analysis of the cis/trans isomerization pathway shows that the mechanisms for the cis/trans isomerization are essentially different between the enol and keto forms.