Search

The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2- aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.

Background Ammonia lyases are enzymes of industrial and biomedical interest. Knowledge of structure-dynamics-function relationship in ammonia lyases is instrumental for exploiting the potential of these enzymes in industrial or biomedical applications. Methods We investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. At this scope, we used microsecond all-atom molecular dynamics simulations, analyzed with dimensionality reduction techniques, as well as in terms of contact networks and correlated motions. Results We identify two regulatory elements in the MAL structure, i.e., the β5-α2 loop and the helix-hairpin-loop subdomain. These regulatory elements undergo conformational changes switching from ‘occluded’ to ‘open’ states. The rearrangements are coupled to changes in the accessibility of the active site. The β5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of tunnels from the protein surface to the catalytic site, making the active site more accessible to the substrate when they are in an open state. Conclusions Our work pinpoints a sequential mechanism, in which the helix-hairpin-loop subdomain of MAL needs to break a subset of intramolecular interactions first to favor the displacement of the β5-α2 loop. The coupled conformational changes of these two elements contribute to modulate the accessibility of the catalytic site. General significance Similar molecular mechanisms can have broad relevance in other ammonia lyases with similar regulatory loops. Our results also imply that it is important to account for protein dynamics in the design of variants of ammonia lyases for industrial and biomedical applications.

14 páginas.-- 10 figuras.-- 1 tabla.-- 46 referencias.-- Author's Choice—Final version free via Creative Commons CC-BY license., Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn2 , and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan, This work was supported by the INDOX (KBBE-2013-613549) European Union project, the HIPOP (BIO2011-26694), NOESIS (BIO2014-56388-R), and BIORENZYMERY (AGL2014-53730-R) projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the PRIN 2009-STNWX3 project of the Italian Ministry of Education, Universities and Research (MIUR)., Supported by an FPI Fellowship of the Spanish MINECO.-- Supported by a contract of the CSIC project 201440E097.-- Supported by a Ramón y Cajal contract of the Spanish MINECO

A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP (versatile peroxidase) expressed in Saccharomyces cerevisiae [García-Ruiz, González-Pérez, Ruiz-Dueñas, Martínez and Alcalde (2012) Biochem. J. 441, 487–498]. 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h of incubation at pH 9 compared with complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. In the present study we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were expressed in Escherichia coli, and their alkaline stability and biochemical properties were determined. In addition, the crystal structures of 2-1B and one of the intermediate variants were solved and carefully analysed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections between haem and helix B (where the distal histidine residue is located), and formation of new electrostatic interactions, that avoided the hexa-co-ordination of the haem iron. These new structural determinants stabilized the haem and its environment, helping to maintain the structural enzyme integrity (with penta-co-ordinated haem iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability., This work was supported by HIPOP [grant number BIO2011-26694], NOESIS [grant number BIO2014-56388-R] and DEWRY [grant number BIO2013-43407-R] projects of the Spanish Ministry of Economy and Competitiveness (MINECO), and by the PEROXICATS [grant number KBBE-2010-4-265397] and INDOX [grant number KBBE-2013-7-613549] European projects. V.S.-J. and F.J.R.-D. are grateful for the financial support of an FPI fellowship and a Ramón y Cajal contract of the Spanish MINECO, respectively.

Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant., We demonstrate that an exposed tryptophan is responsible for high-turnover oxidation by DyP, a representative of a new protein superfamily. Long-range electron transfer from surface tryptophan residues forming radicals appears as a general mechanism for peroxidase oxidation of bulky substrates.

17 p.-2 tab.-8 fig., Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme., This work was funded by the Commission of the European Communities through the INDOX project (KBBE-2013-7-613549, "Optimized oxidoreductases for medium and large scale industrial biotransformations"), and by the Spanish Ministerio de Economía y Competitividad (MINECO) through the HIPOP project (BIO2011-26694, “Screening and engineering of new high-redox-potential peroxidases”).