Carbon Dioxide Utilisation -The Formate Route

UIDB/50006/2020 CEEC-Individual 2017 Program Contract.The relentless rise of atmospheric CO2 is causing large and unpredictable impacts on the Earth climate, due to the CO2 significant greenhouse effect, besides being responsible for the ocean acidification, with consequent huge impacts in our daily lives and in all forms of life. To stop spiral of destruction, we must actively reduce the CO2 emissions and develop new and more efficient “CO2 sinks”. We should be focused on the opportunities provided by exploiting this novel and huge carbon feedstock to produce de novo fuels and added-value compounds. The conversion of CO2 into formate offers key advantages for carbon recycling, and formate dehydrogenase (FDH) enzymes are at the centre of intense research, due to the “green” advantages the bioconversion can offer, namely substrate and product selectivity and specificity, in reactions run at ambient temperature and pressure and neutral pH. In this chapter, we describe the remarkable recent progress towards efficient and selective FDH-catalysed CO2 reduction to formate. We focus on the enzymes, discussing their structure and mechanism of action. Selected promising studies and successful proof of concepts of FDH-dependent CO2 reduction to formate and beyond are discussed, to highlight the power of FDHs and the challenges this CO2 bioconversion still faces.publishersversionpublishe

High-resolution structures of formate dehydrogenase from Candida boidinii.

The understanding of the mechanism of enzymatic recovery of NADH is of biological and of considerable biotechnological interest, since the essential, but expensive, cofactor NADH is exhausted in asymmetric hydrogenation processes, but can be recovered by NAD+-dependent formate dehydrogenase (FDH). Most accepted for this purpose is the FDH from the yeast Candida boidinii (CbFDH), which, having relatively low thermostability and specific activity, has been targeted by enzyme engineering for several years. Optimization by mutagenesis studies was performed based on physiological studies and structure modeling. However, X-ray structural information has been required in order to clarify the enzymatic mechanism and to enhance the effectiveness and operational stability of enzymatic cofactor regenerators in biocatalytic enantiomer synthesis as well as to explain the observed biochemical differences between yeast and bacterial FDH. We designed two single-point mutants in CbFDH using an adapted surface engineering approach, and this allowed crystals suitable for high-resolution X-ray structural studies to be obtained. The mutations improved the crystallizability of the protein and also the catalytic properties and the stability of the enzyme. With these crystal structures, we explain the observed differences from both sources, and form the basis for further rational mutagenesis studies

Biomolecule Arrays Using Functional Combinatorial Particle Patterning on Microchips

Biofunctionalization of surfaces in a microarray format has revolutionized biological assay applications. Here, a microarray system based on a microelectronic chip is presented that allows for a versatile combinatorial in situ molecule synthesis with very high density. Successfully demonstrating an application for peptide array synthesis, the method offers a compact approach, high combinatorial freedom, and, due to the intrinsic alignment, high and reproducible precision. Patterning the chip surface with different microparticle types which imbed different monomers, several thousand different molecule types can be simultaneously elongated layer-by-layer by coupling the particle imbedded monomers to the molecules growing on the chip surface. This technique has the potential for a wide application in combinatorial chemistry, as long as the desired monomeric building blocks are compatible with the chemical process

Peptide Arrays with a Chip

Today, lithographic methods enable combinatorial synthesis of >50,000 oligonucleotides per cm(2), an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. A combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A computer chip consecutively addresses the different charged particles to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids in one single coupling reaction to the support. The method should allow for the translation of entire genomes into a set of overlapping peptides to be used in proteome research

Alternative Setups for Automated Peptide Synthesis

Nowadays, lithographic methods facilitate the combinatorial synthesis of >50.000 oligonucleotides per cm(2), an achievement that revolutionized the whole field of genomics. High-density peptide arrays might spark a similar development for the field of proteomics, but all lithographic methods have a peptide specific disadvantage that impairs their use for peptide synthesis: Each monomer must be coupled separately to the solid support. This adds up to an excessive number of coupling cycles, especially when comparing the 4 x 20 coupling cycles that would generate an array of 20meric oligonucleotides, to the 20 x 20 cycles that would yield an array of 20meric peptides. This review mainly discusses one recent development that leads to very high-density peptide arrays: the combinatorial chemical synthesis based on electrically charged solid amino acid particles. Either a colour laser printer or a chip addresses the different charged amino acid particles to a solid support, where the whole layer of solid amino acid particles is melted. Hitherto immobilized amino acids then start to diffuse to the support, where all the 20 different amino acids couple in a spatially defined manner, and in one single coupling reaction to the support. The method should allow for the translation of entire genomes into sets of overlapping peptides to be used in proteome research

Purification, crystallization and preliminary X-ray study of the fungal laccase from \cal Cerrena maxima

Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 A resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 A (R factor = 18.953%; R(free) = 23.835; r.m.s.d. bond lengths, 0.06 A; r.m.s.d. bond angles, 1.07 degrees) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 A X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO(2) substituents