Periodic Reporting for period 1 - NewCat (Teaching Lytic Polysaccharide Monooxygenases to do Cytochrome P450 Catalysis)
Berichtszeitraum: 2022-06-01 bis 2023-05-31
Activation of sp3-hydridized carbon-hydrogen (C-H) bonds and respective functionalization are central and highly demanding challenges in Nature and also in the chemical industry, due to the high thermodynamic barrier (up to 110 kcal/mol), required selectivity of oxidation, and catalyst stability. The selective C-H activation has been pursued for more than 70 years in all areas of catalysis - homogeneous, heterogeneous and biological - and is considered to be a Holy Grail of chemistry. Nature solves this by the evolution of enzymes containing either iron or copper that use activated oxygen species for selective oxidation of C-H bonds. A central iron containing enzyme is the cytochrome P450 (CYP), a monooxygenase with a heme cofactor. These proteins oxidize steroids, fatty acids, and xenobiotics, and are vital for the clearance of various compounds, as well as for hormone synthesis and breakdown. Hence, CYPs have become essential biocatalysts for industrial production of critical chemicals such as pharmaceuticals and drug metabolites, steroids and antibiotics. CYPs have severe drawbacks, like being membrane-bound and requiring a complex and expensive reducing machinery for their regeneration. An important class of copper containing enzymes was discovered in the past decade by the NMBU’s research team. LPMOs are involved in the oxidative depolymerization of cellulose and chitin, have revolutionized the biorefining industry in the transformation of cellulose to bioethanol. A common feature of CYPs and LPMOs is an active site that provides confinement bringing the oxygen species together with the C-H bond in a "closed" environment lowering the energy of transition states. This allows for a specificity with respect to which C-H bond of the substrate is oxidized. In CYPs, confinement results from a narrow active site buried inside a protein core. In LPMOs, this occurs when the enzyme complexes with the polymeric substrate. Key differences include LPMOs being easy to produce in large scale, small, robust, and rigid water-soluble proteins with a plethora of electron donors. CYPs are membrane bound and require a redox partner such as the cytochrome P450 reductase in combination with NADPH. Moreover, LPMOs have an active site architecture enabling a million-fold higher efficiency in their peroxygenase mechanism versus those of CYPs. This includes conserved amino acids able to “cage” and mediate the conversion of H2O2 to the copper-oxyl intermediate preventing oxidative damage to the enzyme, contrasting CYPs that lack a residue near the active site that can participate in general acid−base catalysis important for the formation of compound I in H2O2-dependent oxidation.
In the NewCat project, we explore to take advantage of creating the necessary tunnel for confinement by immobilizing LPMOs on nanomaterial scaffolds to produce nano-hybrid catalysts that can accommodate substrates of abundance and low-value, or an intermediate in a reaction chain, to yield products of high-value. This new catalytic system is expected to have a number of advantages and unique features: (i) enzymes immobilized on nanomaterial scaffolds are more robust, stable, and recoverable than free entities; (ii) reaction speed-up due to high/efficient enzyme concentration; (iii) the high oxidative power provides opportunities to design a versatile technology for valorization of several classes of natural compounds, including components of the highly available lignin; and (iv) environmentally beneficent processes are possible, such as organic and plastic polymers waste decomposition.
In the NewCat project, we explore to take advantage of creating the necessary tunnel for confinement by immobilizing LPMOs on nanomaterial scaffolds to produce nano-hybrid catalysts that can accommodate substrates of abundance and low-value, or an intermediate in a reaction chain, to yield products of high-value. This new catalytic system is expected to have a number of advantages and unique features: (i) enzymes immobilized on nanomaterial scaffolds are more robust, stable, and recoverable than free entities; (ii) reaction speed-up due to high/efficient enzyme concentration; (iii) the high oxidative power provides opportunities to design a versatile technology for valorization of several classes of natural compounds, including components of the highly available lignin; and (iv) environmentally beneficent processes are possible, such as organic and plastic polymers waste decomposition.
We have established a consortium that is excellently equipped to produce a universal platform for the selective hydroxylation of sp3- hybridized C-H bonds. The internal communication of progress and results and decision making of what next steps to take are well founded. So far, we have access to ~35 lytic polysaccharide monooxygenases (LPMOs), either newly cloned or taken from our NMBU library, that varies greatly in substrate preference, hydrophobicity in the active site, and topology. This gives a vast pool of LPMOs to choose from with respect to the new substrates that are to be functionalized. Moreover, we have 18 functionalized carbon nanotubes (CNT) providing differences in charge, polarization, as well as distance from the support to the active site allowing accommodation of the substrates of interest. Preliminary results show that LPMOs strongly adhere to several of the CNTs. Furthermore, the LPMOs retain catalytic activity while being immobilized. Calculations show that the innate active site of LPMOs are capable of performing hydroxylation of desired positions in our substrates, i.e. monolignols and steroids. We have also established models depicting the chemical determinants of the adherence of LPMOs with carbon-based support, providing a valuable tool for future loop designs for LPMOs. Moreover, we have developed an mass spectrometry based method allowing for the qualitative and quantitative analysis of hydroxylated monolignols. Using this method, we have detected innate LPMO activity on these substrates. As benchmarks, cytochrome P450 BM3 and selected unspecific peroxygenases also show specific hydroxylation of monolignols. We are also establishing a nuclease-based in-vivo mutagenesis tool using CRISPR allowing for the controlled change of residues in the active site as well as for loops modification. Preliminary up-scaling of LPMO production show that 0.5 g of protein can be produced from a 5 L fermentation.
To our knowledge, the demonstration of adherence of LPMOs on CNTs while remaining active has not yet been shown before our work. This alone shows proof-of-concept for the whole project. Moreover, we demonstrate that native LPMOs have innate activity on monolignols and a steroid substrate even though these are not natural substrates for the enzymes. Calculations demonstrate that the proposed reactive intermediate in LPMO catalysis, Cu(II)-oxyl, harbor sufficient hydroxylating power to activate C-H bonds of desire. At NMBU, we have gained an enormous amount of knowledge on role of amino acid residues near the active site, dubbed second sphere residues, have on Cu(II/Cu(I) reactivity as well as controlling the fate of the reactive intermediate. This coupled with our developed in silico analysis to assess individual amino acid interaction with the monolignols and steroids, we have a powerful tool to predict what alterations needed to take place for optimal binding of the non-natural substrates.