Computational analysis of mechanism and substrate specificity of an ADH from Candida parapsilosis

Carbonyl reductase 2 from Candida parapsilosis (CPCR2) is one of the promising candidate amongst the zinc-dependent medium chain dehydrogenases/reductases (MDRs) for the biocatalytic production of enantiomeric pure alcohols from their corresponding aldehyde/ketones. The enantiomeric chiral alcohols act as important building blocks for chemical and pharmaceutical industries. In this thesis, we aim to investigate the carbonyl reduction mechanism and substrate specificity of CPCR2. Initially, the catalytic and resting state of CPCR2 were identified and further carbonyl reduction mechanism catalyzed by MDRs were studied using a multiscale approach combining quantum mechanics and molecular mechanics (QM/MM) simulations. The catalytic zinc environment of CPCR2 was re-parameterized to have suitable partial charges on zinc ion and coordinated residues (Cys44, His65, and Asp154) for the development of a substrate based docking protocol. The identified catalytic state was used for the development of a substrate based docking protocol including catalytic constraints for carbonyl substrates in the zinc-dependent MDRs. The developed docking protocol was further validated against experimentally determined kinetic parameters and a correlation of 0.73 was achieved allowing for the first time predictive docking of ADH substrates. Our cooperative partner experimentally identified three amino acid positions (Cys57, Trp116, and Leu119) of CPCR2 which contributes to the substrate specificity in CPCR2. These positions were explained using flexible docking protocol and found that these are responsible for widening the binding pocket in CPCR2. In the last part of this thesis, we have performed in silico study about substrate scope of CPCR2 using substrate docking based virtual screening of 16713 carbonyl substrates from the ZINC database. Afterwards, KEGG and Candida Genome pathway search was performed to identify the natural substrates for CPCR2 and identified butyraldehydes as a potential natural substrate of CPCR2, suggesting carbonyl reductase role for this enzyme in butyraldehyde reduction. Phylogenetic tree shows that CPCR2 is more close to ADH5 from Candida alibicans with identity of 74.11 % and we therefore propose the correct nomenclature for CPCR2 should be butyraldehyde dehydrogenase with a standard name as cpADH5. In conclusion, the identified structure-function relationships and predictive computational tools enabled the rational engineering of zinc-dependent ADH and offers novel and exciting opportunities to reengineered CPCR2 and other zinc-dependent ADHs into industrially attractive enzymes