Phylogenetic and Structural Analysis of Polyketide Synthases in Aspergilli

Abstract

Polyketide synthases (PKSs) of Aspergillus species are multidomain and multifunctional megaenzymes that play an important role in the synthesis of diverse polyketide compounds. Putative PKS protein sequences from Aspergillus species representing medically, agriculturally, and industrially important Aspergillus species were chosen and screened for in silico studies. Six candidate Aspergillus species, Aspergillus fumigatus Af293, Aspergillus flavus NRRL3357, Aspergillus niger CBS 513.88, Aspergillus terreus NIH2624, Aspergillus oryzae RIB40, and Aspergillus clavatus NRRL1, were selected to study the PKS phylogeny. Full-length PKS proteins and only ketosynthase (KS) domain sequence were retrieved for independent phylogenetic analysis from the aforementioned species, and phylogenetic analysis was performed with characterized fungal PKS. This resulted into grouping of Aspergilli PKSs into nonreducing (NR), partially reducing (PR), and highly reducing (HR) PKS enzymes. Eight distinct clades with unique domain arrangements were classified based on homology with functionally characterized PKS enzymes. Conserved motif signatures corresponding to each type of PKS were observed. Three proteins from Protein Data Bank corresponding to NR, PR, and HR type of PKS (XP_002384329.1, XP_753141.2, and XP_001402408.2, respectively) were selected for mapping of conserved motifs on three-dimensional structures of KS domain. Structural variations were found at the active sites on modeled NR, PR, and HR enzymes of Aspergillus. It was observed that the number of iteration cycles was dependent on the size of the cavity in the active site of the PKS enzyme correlating with a type with reducing or NR products, such as pigment, 6MSA, and lovastatin. The current study reports the grouping and classification of PKS proteins of Aspergilli for possible exploration of novel polyketides based on sequence homology; this information can be useful for selection of PKS for polyketide exploration and specific detection of Aspergilli.

Keywords: Aspergillus; ketosynthase; phylogeny; polyketide; polyketide synthases.

Figure 1

PKS genealogy of Aspergillus PKSs with characterized PKS (1000 boot strap value). Notes: Genealogy of KS domain from type I PKSs of Aspergillus species is inferred by maximum parsimony analysis of the Aspergillus PKSs. Major clades and subclades are indicated by vertical bars that share a common organization of domains. Designations beginning with Afu, Afl, Ani, Atr, Aor, Apr, and Acl correspond to the A. fumigatus, A. flavus, A. niger, A. terreus, A. oryzae, A. parasiticus, and A. clavatus, respectively. Details of these PKS can be found in Supplementary Table 1. Protein FASs from G. gallus, H. sapiens, Caenorhabditis briggsae, and C. elegans served as an outgroup for this study. Bar colored in blue represents NR PKS with the domain architect SAT-KS-AT-ACP-ACP-TE/PP. Bar colored in green shows PR PKS with the domain architects KS-AT-ACP-MT-TE/PP. Bar with dark green color shows HR PKS with the domain architect KS-AT-DH-MT-ER-KR-TE/PP and also other HR domain containing PKS, such as KS-AT-DH-MT-KR-TE/PP and KS-AT-DH-MT-KR-ER-TE/PP. Hybrid PKS–NRPSs are noted with bar colored in orange with the domain architect of both PKS and NRPS: KS-AT-DH-MT-ER-KR-ACP-ACP-C-A-PCP-DKC.

Figure 2

KS genealogy of Aspergillus PKSs (1000 boot strap value). Notes: Phylogeny of KS domain from type I PKSs of Aspergillus species is inferred by maximum parsimony analysis. Major clades and subclades are indicated by vertical bars, which share a common organization of domains. Numbers below branches indicate percentage bootstrap values for each clade. KS domain from A. flavus, A. fumigatus, and A. niger sequenced in this study is also included, and details of which can be found in Supplementary Table 1.

Figure 3

Conserved motifs on type I KS sequences. Notes: Conserved motifs observed in the type I KS for Fungal PKSs are presented in the table.

Figure 4

Three-dimensional modeled structures of NR, PR, and HR type of KS. Notes: Structures have been modeled for KS domain from protein designated as 6MSAS producing 6-mehtlysalisylic acid (PR), PKS P producing pigment (NR), and LNKS producing lovastatin (HR). The cavities of the modeled structures have been shown in surface rendering and circled in red. Each model has been superimposed with the structural template of 1KAS.

Figure 5

Three-dimensional modeled structure from Aspergillus KSs. Notes: NR type KS Afl25 (XP_002384329.1) predicated to produce pigment, HR type KS Afu1 (XP_753141.2) predicated for lovastatin, and PR type KS Anr (XP_001402408.2) predicated for 6-metylsalicylic acid were modeled using the template structure of beta-ketoacyl-ACP synthase II from Escherichia coli (PDB ID: 1KAS). Tyr and Ala residues have been marked on the surface topology. Catalytic triad Cys-His-His are also marked in the modeled structure for validation in stick model in different colors, namely, purple, green, and blue.