Research Highlights
In our laboratory a multidisciplinary approach combining Biochemistry, Microbiology, Molecular Biology, Protein Engineering, and Structural Biology has been followed to:
(i) advance the understanding of the catalytic, stability and structural fingerprints of bacterial oxidoreductive enzymes potentially involved in xenobiotics degradation and lignocellulose conversion, (ii) improve their performance and robustness using protein engineering approaches, including directed evolution, (iii) identify intermediates and products of enzymatic reactions through complementary information derived from Nuclear Magnetic Resonance (NMR) and Mass Spectrometry, (iv) use the acquired knowledge and tools to the set-up of multi(-step)-enzymatic or chemo-enzymatic green processes for the production of added-value compounds.
We are pioneers in structural and functional characterisation of bacterial laccases. Laccases show the broadest substrate specificity among the ligninolytic enzymes, extended to substituted phenol, polyphenols, aromatic amines, and thiols, requiring only atmospheric oxygen as co-substrate. Important insights were given on the role of synthetic and phenolic redox mediators that enhanced the substrate range of these biocatalysts. A toolbox of experimental approaches was mounted at ITQB to explore these biocatalysts not only for the detoxification of industrial synthetic dyes but also for degradation of non-phenolic lignin units. Efficient expression systems to produce large quantities of these enzymes and tools to genetically manipulate them were developed. Thorough multidisciplinary investigations of wild type and engineered variants revealed key functional aspects of these enzymes, such as the solvent accessibility of the catalytic centres, the electrostatic interactions modulating the redox potential and the molecular mechanisms behind their thermodynamic stability. In particular, the role of of key residues in the active site(s) of CotA was established paving the way to manipulate its redox properties (i.e. range of oxidizing substrates) or substrate specificity in a desired fashion.
The studies of laccase-like enzymes from hyperthermophilic microorganisms provided the first evidence that these enzymes possess notable metal oxidase activity and an extreme intrinsic thermostability, which is worthwhile exploring for engineering of enzymes with improved chemical robustness. A laboratory evolution approach was conducted to improve the efficiency of the metallo-oxidase McoA from the hyperthermophilic bacterium Aquifex aeolicus for aromatic compounds. Four rounds of random mutagenesis of the mcoA-gene followed by high-throughput screening (≈ 94,000 clones) led to the identification of the 2B3 variant featuring a 2-order of magnitude higher efficiency than the wild-type enzyme for the typical laccase substrate ABTS (2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)) and showing additionally a higher activity for phenolics and synthetic aromatic dyes. Notably, the recombinant 2B3 variant, unlike the wild-type, does not show temperature and chemical-dependent aggregation, exhibiting an enhanced solubility and thus a higher kinetic and thermodynamic thermostability.
Enzymatic conversion of aromatic amines into valuable compounds, such as phenazines and phenoxazinone derivatives, indo dyes and azobenzene dyes using the CotA-laccase enzyme was established. For example, phenazines and phenoxazinones are heterocyclic nitrogen containing compounds that are broadly distributed in natural and synthetic products and are active motifs of antibiotics, anti-tumor agents, pesticides, dyestuffs, biosensors. The ability of CotA-laccase to mediate coupling reactions leading to the synthesis of these compounds was demonstrated using several m,p-disubstituted phenylamines, o-phenyldiamines, substituted o-aminophenols substrates and o-substituted diamines. Overall, the CotA-laccas enzymatic-catalysed sequences were shown to constitute a valuable alternative to the chemical oxidative coupling of aromatic amines precursors for the production of different heterocyclic scaffolds.
We have recently focused on bacterial dye-decolourising peroxidases (DyP-type), novel enzymes that have primary sequence, structural and apparently mechanistic features unrelated to those of other known peroxidases. They successfully degrade a wide range of dyes, including anthraquinone-based and azo dyes, and non-phenolic lignin units in the absence of redox mediators. It was demonstrated that DyPs from different sources reveal distinct stability, substrate specificity, catalytic efficiency and redox properties, as a basis for exploring their potential to replace the high-redox fungal lignin and versatile peroxidases in biotechnological applications. We have elucidated for the first time the catalytic mechanism of a DyP showing that the reaction of BsDyP with H2O2 exhibits saturation behaviour consistent with a two-step mechanism involving the formation of an E-H2O2 intermediate, the so–called Compound 0, followed by formation of Compound I. Interestingly, even if this result was predicted in the Poulos-Kraut model, it was only previously observed in very few kinetic studies at low temperatures.
Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by error-prone PCR of the ppDyP-gene followed by high-throughput screening allow identifying 6E10 variant showing 100-fold enhanced catalytic efficiency for phenolic lignin-related substrate, 2,6-dimethoxyphenol (DMP). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin and the lignin phenolic model dimer, guaiacylglycerol-β-guaiacyl-ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units as compared to the wild-type, showed resistance to hydrogen peroxide inactivation, and was produced at 2-fold higher yields. The acquired mutations in the course of the evolution affected three amino acid residues (E188K, A142V and H125Y) situated at the surface of the enzyme, in the second shell of the heme cavity. Biochemical analysis of hit variants from the laboratory evolution, and single variants constructed using site-directed mutagenesis, unveiled the critical role of acquired mutations from the catalytic, stability and structural viewpoints. We show that epistasis between A142V and E188K mutations is crucial to determine substrate specificity of 6E10. Evidence suggests that ABTS and DMP oxidation occurs at the heme access channel.
