Investigating the Catalytic Mechanisms of Bio-degrading Copper Proteins: Multi-copper Oxidases (MCOs) and Lytic Polysaccharide Monooxygenases (LPMOs)
<p>Lignin and cellulose comprise a large portion of the renewable biomass on Earth. However, substantially due to laborious course of processing, the conversion efficiency of these biomaterials to accessible biofuel is very low. Therefore, effective depolymerization and utilization of these bi...
| Summary: | <p>Lignin and cellulose comprise a large portion of the renewable biomass on Earth. However, substantially due to laborious course of processing, the conversion efficiency of these biomaterials to accessible biofuel is very low. Therefore, effective depolymerization and utilization of these biopolymers are requirements for environmentally friendly and sustainable energy development. In the hope of finding solutions to these biomass utilization challenges, there have been growing interests in using biodegrading metalloenzymes as active biocatalysts. However, there still remain many questions regarding mechanistic details of enzyme catalysis and effective application of these enzymes. This thesis focuses on investigating the redox chemistry involved in the catalytic mechanisms of two main lignin- and cellulose- degrading copper enzymes: multicopper oxidases (MCOs) and lytic polysaccharide monooxygenases (LPMOs).</p>
<p>MCOs are capable of aerobic oxidation of lignin as their primary function, but the nature of their substrate variability also allows the oxidation of not only diverse high potential organic and inorganic complexes, but also earth abundant divalent metal ions such as manganese. LPMOs, on the other hand, enable the cleavage of glycosidic bonds in recalcitrant insoluble cellulosic substances, which are not degradable by other hydrolytic enzymes such as endoglucanases and cellulobiohydrolases.</p>
<p>It is remarkable that nature has created such versatile enzymes with specific active site metals and redox-active amino acids involved in electron transfer, which contribute to substrate oxidation as well as enzyme survival against oxidative damage during catalysis. By gaining a deeper understanding of how these enzymes work, we could greatly enhance current usage efficiencies and develop more energy-efficient biocatalysts.</p>
<p>Chapter I gives an introduction to biological coppers, two groups of bio-degrading copper enzymes: multicopper oxidases (MCOs) and lytic polysaccharide monooxygenases (LPMOs), and the role of redox-active amino acids in electron transfer and enzyme catalysis. For the MCO work, a thermophilic laccase (<i>Tth</i>-lac) from <i>Thermus thermophilus</i> HB27 and a CotA laccase (CotA-lac) from <i>Bacillus Subtilis</i> were studied. For the LPMO work, two cellulose active LPMOs (ScLPMO10B and ScLPMO10C) and a chitin active LPMO (BlLPMO10A) were studied.</p>
<p>Chapter II describes thermodynamic aspects of <i>Tth</i>-lac catalysis. The temperature dependence of the formal potential of type I copper (Cu<sub>T1</sub>) in <i>Tth</i>-lac is reported, and the interplay between many competing dynamic and thermodynamic factors which results in thermostability and activity of <i>Tth</i>-lac is discussed.</p>
<p>Chapter III reports the electron transfer (ET) kinetics data obtained with <i>Tth</i>-lac using the transient absorption spectroscopy. The results of photochemical electron/hole transfer studies indicate that the chains of Trp and Tyr can participate in electron transfer through <i>Tth</i>-lac, which could potentially have a role in enzyme catalysis as well.</p>
<p>Chapter IV discusses the protective role of a Trp/Tyr pair positioned close to the trinuclear copper cluster (TNC) in <i>Tth</i>-lac. It is indeed remarkable that laccases are capable of utilizing the power of oxygen to catalyze the oxidation of diverse high-potential substrates. But, as a tradeoff, the utilization of dioxygen can make the enzyme highly susceptible to oxidative damage. Chapter IV provides supporting evidence that led us to conclude that the TNC-proximal Trp/Tyr pair functions as an internal antioxidant for prolonging the enzyme lifetime.</p>
<p>Chapter V describes investigations on the factors that affect MCO catalysis, which include the potentials of the active site coppers, possible reactive intermediates, and common structural motifs. Based on the structural homology between <i>Tth</i>-lac and CotA-lac, some preliminary work done on CotA-lac is also reported.</p>
<p>Chapter VI outlines the work on LPMOs. After the successful expression and purification of ScLPMO10B, ScLPMO10B and BlLPMO10A, standard activity assays were done with insoluble cellulose and chitin substrates to confirm the enzyme activity. The results are compared with that from the photo-degradation experiments to investigate if the photochemically generated Cu(III) species are active intermediates in LPMO catalysis.</p>
<p>Chapter VII reports the results on bioinformatics analysis on the distribution of vicinal amino acids in different enzyme classes. This study was to examine the biological significance of amino acid pairs and clusters existing in many different enzyme classes, with vicinal surface tyrosines in CotA-lac as an underlying motivation behind the work.</p>
<p>This thesis demonstrates that MCOs and LPMOs are truly versatile enzymes which can oxidize such diverse refractory substrates, and there could be multiple pathways that the enzymes achieve this task. As shown so far, not only the active site metals but also the chain of redox-active amino acids as well as metal coordinating residues can contribute to enzyme catalysis.</p> |
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