Phylogenetic And Structural And Functional Analysis Of Cytochrome P450 Monooxygenase CYP5619A1 From Saprolegnia Diclina

Abstract

Genome sequencing of lower eukaryotes such as fungi revealed high diversity of cytochrome P450 monooxygenases (P450s/CYPs) in their genomes compared to other biological kingdoms. For example, not only the presence of a large number of P450s was detected in many of their genomes, but also high diversity in terms of the number of P450 families. P450s are heme-thiolate proteins distributed across the biological kingdoms with immense catalytic diversity, which has prompted the use of these enzymes as potential catalysts for the production of fine chemicals, pharmaceutical compounds, antibiotics, fragrances and detoxification of carcinogenic and/or mutagenic compounds. Progress has been made in understanding P450s from lower eukaryotic organisms, which has led to the unravelling of their potential as anti-fungal drug targets. The lower eukaryotes belonging to the kingdom Stramenopila, especially phylum Oomycota species P450s, have been underexplored. Oomycetes are “hard-wired parasites” that remain a serious problem in agriculture and aquaculture and are counted among the most widespread and deadliest disease-causing agents of plants and crops worldwide. Their destructive behaviour lies in their ability to breach the host surface and break it down, promptly resulting in extensive destruction that hinders agricultural growth. The impact of oomycete species on the economy triggered various investigations on pathogenesis and control methods for these pathogens. In the quest to find a remedy, genome sequencing of oomycetes was carried out. Recently, the Unit for Drug Discovery Research’s laboratory (Department of Health Sciences, Faculty of Health and Environmental Sciences at the Central University of Technology) performed comprehensive comparative P450 genomics in 13 oomycete pathogens and discovered six novel P450s that can be used as drug targets against these pathogens, particularly fish pathogens. The novel P450s belong to the CYP5619 family and were found in the fish pathogen Saprolegnia diclina. In order to use these P450s as novel drug targets, it is of the utmost importance to perform biochemical and biophysical characterisation of the family members. Hence, I am herewith proposing to perform in silico structural and functional analysis of CYP5619A1 from S. diclina, including cloning and generation of recombinant E. coli cells containing the CYP5619A1 gene in a novel expression vector and comprehensive phylogenetic analysis. This study is the first of its kind on analysis of the novel P450 family CYP5619 in microbes. Phylogenetic analysis of CYP5619 family members across biological kingdoms revealed the presence of this novel P450 family in other oomycetes and in a phytoplankton. However, the number of CYP5169 members in organisms varied. Nine CYP5619 members were found in Achlya hypogyna and six were found in S. parasitica (both oomycetes). The oomycetes, Thraustotheca clavata and Aphanomyces invadans, were found to have three and two CYP5619 members, respectively. Emiliania huxleyi, a phytoplankton, was found to have two CYP5619 family members, but the smallest count was attributed to an oomycete, A. astaci (one CYP5619 member). This suggests that the CYP5619 family is present in other organisms apart for oomycetes. After performing phylogenetic analysis, a 3D model of CYP5619A1 from S. diclina was built by homology modeling and assessed for its binding affinity with different predicted substrates and with malachite green, a remedy used to treat S. diclina infections. The study revealed that eight of the compounds required low energy to bind to the target protein, with binding energies below -6.00 kcal/mol. This suggests that these ligands can act as possible substrates of CYP5619A1. Among all ligands, linoleic acid and malachite green showed a high binding affinity with the CYP5619A1 model. Linoleic acid is a polyunsaturated fatty acid with 18 carbon atoms and two double bonds in its structure. Malachite green is an organic compound that is widely used in aquaculture to treat S. diclina infections. These two compounds appeared to be the compounds with the best affinities to the target protein. In this regard, it is reasonable to believe that linoleic acid-like compounds could be potential substrates for CYP5619A1 and malachite green possibly inhibiting CYP5619A1 in S. diclina. In order to validate in silico results, CYP5619A1 was cloned into the newly designed vector pINK-d using in silico and in vitro techniques. The gene was cloned into the novel expression vector using the software pDRAW and the sequence of the designed primers, vector and gene of interest were sent to GenScript. The vector and vector with CYP5619A1 obtained from GenScript were subjected to restriction enzyme analysis. The results obtained were satisfactory, as the gene was perfectly cloned into the vector, which was verified by running the plasmid through an agarose gel on the one hand, and the plasmid restricted by the KpnI and XbaI enzymes on the other hand. The gel analysis confirmed the presence of CYP5619A1.