Identification,Classification,and Curation Of Novel Unspecific Peroxygenases

Unspecific peroxygenase(UPO)(EC 1.11.2.1)is an extracellular enzyme secreted by fungal species.This enzyme came into existence more than a decade ago and represents an intriguing oxidoreductase sub-subclass of heme proteins with a peroxygenase and peroxidase activity.With more than 300 identified substrates,UPOs are capable of catalyzing a wide range of oxidation reactions such as 1-or 2-electron oxygenation,selective oxyfunctionalizations,epoxidation,sulfoxidation,aromatization,halide oxidation.Being most efficient in peroxygenation makes them most significant in organic syntheses and potentially attractive as industrial biocatalysts.There exists a very few identified UPOs with distinct properties,notably,Marasmius rotula UPO(MroUPO)shows behavior ranging between UPO and another heme-thiolate peroxidase,called Chloroperoxidase(CPO).The intriguing characteristics and catalyzing functions of UPOs prompted us to search for more UPOs in the fungal kingdom.This led us to study their evolutionary relationship with CPOs.This work is focused on finding novel UPOs,study their phylogeny,evolution,and organize them by a proper classification system by developing a database dedicated to UPO encoding protein sequences.Firstly,an analysis pipeline was developed to search and identify novel UPOs from more than 800 fungal genomes available in the Ensembl database at the time of the study.This pipeline consists of various filters including homology search,sequence-based,and graph-based clustering,and motif search.These filters helped to remove redundant and non-UPO sequences.This pipeline is applicable to any enzyme with a known motif pattern.In this study,we used this pipeline to search for more UPO encoding protein sequences from thousands of genome peptide sequences present in the Ensembl Fungi database.Secondly,in this study,novel UPOs in fungal genomes were searched and 113 putative UPOencoding protein sequences were found distributed in 35 different fungal species(or strains),amongst which single sequence per species was subjected to phylogeny study along with CPOs.The phylogenetic study shows that the UPOs are distributed only in Basidiomycota and Ascomycota phyla of fungi.Sequence analysis helped to classify the UPOs into five distinct subfamilies: classic AaeUPO and four new subfamilies with potential new traits.This study has also shown that each of these five subfamilies(supported by)have their own signature motifs.Surprisingly,some of the CPOs appeared to be a type of UPOs indicating that they were previously misidentified.Further,the obtained UPOs and CPOs were subjected to selection pressure and functional divergence analyzes to study their relationship with each other and CPOs.Selection pressure was observed on important motifs in UPOs which could have driven their functional divergence.Furthermore,the sites having different evolutionary rates caused by the functional divergence were also identified on some motifs along with the other relevant amino acid residues.Finally,critical amino acids responsible for the functional divergence in the UPOs were predicted.Some sequence differences of MroUPO were identified with respect to UPOs and CPOs to predict it's ranging behavior.These results provide novel UPO-encoding sequences,a glimpse of their evolution from CPOs,and present new insights on their functional divergence.A new classification of UPOs has been presented in this study and shed new light on its phylogenetics.Thirdly,more UPOs were searched using the pipeline providing thousands of UPO-encoding sequences,which are difficult to analyze without proper organization and therefore,an online database was created called Unspecific Peroxygenase Database(UPObase).UPObase(upobase.bioinformaticsreview.com)currently includes 1948 peroxygenase encoding protein sequences mined from fungal genomes.It provides information about each sequence including classification,motifs,new sequence submission portal,homology search against UPObase,and sequence analyses such as multiple sequence alignments(MSA)and phylogenetic trees.The database has been made user-friendly facilitating systematic search and filters.UPObase allows users to search for the sequences by organism name,cluster ID,and accession number.Notably,the previously proposed classification system remains established.In this study,using 1948 UPOs in the database,six novel sub-superfamilies(Pog-a,Pog-b,Pog-c,Pog-d,Pog-e,and Pog-f)with signature motifs and two distinct groups in Subfamily-I and III,Ia and Ib,IIIa and IIIb respectively were further identified.