An improved and general streamlined phylogenetic protocol applied to the fatty acid desaturase family

Abstract

Numerous tools to generate phylogenetic estimates are available, but there is no single protocol that will produce an accurate phylogenetic tree for any dataset. Here, we investigated some of those tools, paying particular attention to different alignment algorithms, in order to produce a phylogeny for the integral membrane fatty acid desaturase (FAD) family. Herein, we report a novel streamlined protocol which utilises peptide pattern recognition (PPR). This protocol can theoretically be applied universally to generate accurate multiple sequence alignments and improve downstream phylogenetic analyses. Applied to the desaturases, the protocol generated the first detailed phylogenetic estimates for the family since 2003, which suggested they may have evolved from three functionally distinct desaturases and further, that desaturases evolved first in cyanobacteria. In addition to the phylogenetic outputs, we mapped PPR sequence motifs onto an X-ray protein structure to provide insights into biochemical function and demonstrate the complementarity of PPR and phylogenetics.

Consensus mutagenesis and ancestral reconstruction provide insight into the substrate specificity and evolution of the front-end Δ6-desaturase family

“…7, Supplementary Table 2). As Δ6desaturases, including MpΔ6des, cannot be readily purified in an active form, 55 whole-cell measured activity values (triplicate) were normalized against a wild-type MpΔ6des control included in every set of assays. The level of Δ6-desaturase expression was determined using…”

Peptide Pattern Recognition for high-throughput protein sequence analysis and clustering

“…PPR version 2 is a fast method for dividing large families of protein sequences into related groups to facilitate phylogenetic analysis (Wilding et al, 2017) and functional sequence analysis (Agger et al, 2017;Lange, 2015, 2013). The lists of conserved, short sequences provided for each group can be used for structure-function investigations and for annotation of proteins e.g.…”

“…Recently, PPR was used to divide the integral membrane fatty acid desaturase family into groups of sequences that could be used for phylogenetic analysis (Wilding et al, 2017). However, only a limited number of the sequences could be grouped due to low processing capacity of the previously published PPR software.…”

Integral Membrane Fatty Acid Desaturases: A Review of Biochemical, Structural, and Biotechnological Advances

“…The first overall phylogenetic analysis of the DES family was published in 2003 by Sperling et al [34] and was recently updated. [123] The phylogeny of the DES family shows a distinctive separation of the enzymes by function rather than organism, with some distant members sharing sequence similarity of only 10% [34] (Figure 8). DES are generally broadly distributed through all kingdoms of life apart from Archaea.…”