Configurational Assignment of Secondary Hydroxyl Groups and Methyl Branches in Polyketide Natural Products through Bioinformatic Analysis of the Ketoreductase Domain

Abstract

Profile hidden Markov models (HMMs) were used to predict the configuration of secondary alcohols and α-methyl branches of modular polyketides. Based on the configurations of two chiral centers in these polyketides, 78 ketoreductases were classified into four different types of polyketide producers. The identification of positions that discriminate between these protein families was followed by fitting six profile HMMs to the data set and the corresponding subsets, to model the conserved regions of the protein types. Ultimately, the profile HMMs described herein predict protein subtypes based on the complete information-rich region; consequently, slight changes in a multiple sequence alignment do not significantly alter the outcome of this classification method. Additionally, Viterbi scores can be used to assess the reliability of the classification.

Evaluating Ketoreductase Exchanges as a Means of Rationally Altering Polyketide Stereochemistry

“…We therefore sourcedK Rd omains from modules of this type, ac hoice which additionally simplified the definition of the domain boundaries (the boundaries are shown in Figure S1 in the Supporting Information). We obtained and cloned 14 such epimerizing KR domains: [26] eight A2-type KRs (amphotericin (Amph) KR 1 and KR 11 , [27] concanamycin (Con) KR 4 and KR 10 , [28] elaiophylin (Ela) KR 4 , [29] oligomycin (Olm) KR 5 , [30] pimaricin (PIMS) KR 7 , [31] and candicidin (Can) KR 13 ) [32] and six B2-type KRs (erythromycin (DEBS) KR 1 , [33] lankamycin (Lkm) KR 1 , [34] pikromycin (Pik) KR 1 , [35] lasalocid (Las) KR 7 , [36] ECO-02301 (ECO) KR 19 , [37] and stambomycin (Sam) KR 21 ). [38] For KRs that are preceded by ar ecently identified dimerizationd omain, [39] the sequence encoding this region was also included, as done previously.…”

Biological characterization of the hygrobafilomycin antibiotic JBIR-100 and bioinformatic insights into the hygrolide family of natural products

“…To corroborate this assignment and to furnish the absolute configuration, we bioinformatically analyzed several of the ketoreductase (KR) domains. McDaniel and Caffrey previously determined key sequence signatures that separated KR domains into A- and B-type [78, 79], and this approach has been further expanded by more sophisticated methods including hidden Markov model (HMM)-based sequence classification [80]. This sequence-based approach has been used successfully to assign the absolute configuration of several polyketides and has been corroborated by analysis of the crystal structures of several representative KRs [14, 81–83].…”

“…S9, Supplementary data). Kitsche and Kalesse demonstrated that KR sequence could also occasionally distinguish methyl stereochemistry [80]; indeed, using their HMM-based classification program ScoreDiff, KR9 is confidently predicted to give rise to an l α-methyl branch, although KR2, KR4, and KR5 methyl stereochemistry could not be reliably predicted using this method. The KR9 stereospecificity classification is especially important in the context of the spectroscopic ambiguity of the 8-C stereocenter.…”

“…KR sequences were aligned using MUSCLE (v. 3.8.31, http://www.ebi.ac.uk/Tools/msa/muscle/) on default settings [101, 102]. Classification was performed by the absence or presence of diagnostic motifs identified by previous studies as well as by use of the ScoreDiff web tool (https://akitsche.shinyapps.io/profileHMM_App) [78–80]. Alignments with indicated motifs are given in Fig.…”

Die Pyxipyrrolone: Strukturaufklärung und Biosynthese zytotoxischer myxobakterieller Sekundärmetabolite

“…Dies würde nicht nur den Alkohol an C-27 erklären, fürd en das Vorhersageverfahren von Kitsche und Kalesse die richtige Ste-reochemie prognostiziert, [12] sondern auch die in Modul 4e rzeugte Doppelbindung. Dies würde nicht nur den Alkohol an C-27 erklären, fürd en das Vorhersageverfahren von Kitsche und Kalesse die richtige Ste-reochemie prognostiziert, [12] sondern auch die in Modul 4e rzeugte Doppelbindung.…”