New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase, PKSIIINc in Neurospora crassa

Abstract

Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIII, a functionally and structurally characterized type III PKS from the fungus, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIII through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIII catalysis. Site-directed mutagenesis of 120 and186 to and, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIII product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIII products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of 120 to bulky side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIII protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.

Identification of Stress-Related Genes and a Comparative Analysis of the Amino Acid Compositions of Translated Coding Sequences Based on Draft Genome Sequences of Antarctic Yeasts

“…Type III PKSs are more rarely described in fungi. At present, ten of these PKSs have been reported from Ascomycota and only one from Basidiomycota, and they play several physiological roles in such processes as pigmentation, salinity and dehydration resistance, stress adaptation, and cell wall remodeling ( Parvez et al, 2018 ; Navarro-Muñoz and Collemare, 2019 ). To the best of our knowledge, no reports have described this kind of secondary metabolite cluster in C. sake and Cryptococcus sp.…”

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis

“…The equivalent position in PKSIII of N. crassa (position 120) is a key residue in determining enzymatic function. It has been shown that substitution of a C120 by a phenylalanine or serine in PKSIIINc results in synthesis of phloroglucinol instead of α-pyrone [50]. The active site is flanked by the acyl-binding tunnel similar to those observed in the structures of bacterial and fungal PKSIII [38,39,47,50,53].…”

“…The modelling revealed that this protein could form a dimer complex, like other fungal PKSIII ( Figure 2 A). We compare PKSIII Mo29 amino acids involved in substrate binding with those found in PKSIII from PKSIII from N. crassa (PKSIIINc) [ 37 , 47 , 50 ], Mycobacterium tuberculosis (PKS18) [ 51 ], two PKSIII from Aspergillus oryzae (AoCsyA and AoCsyB) [ 38 , 39 , 52 ], PKSIII from Botrytis cinerea (BcPKS) [ 53 ] and PKSIII from Aspergillus niger (AnPKS) [ 41 ]. The active site residues cysteine 171, histidine 351 and asparagine 385 (catalytic sites) are conserved ( Table 1 and Figure 2 B) ( Figure S2 ).…”

“…In addition, residues involved in the cyclisation of malonyl-CoA were also conserved (cysteine 139 and threonine 140) ( Figure 2 B) [ 50 ]. Previous work has demonstrated that serine at position 186 is essential for condensation of aldols in PKSIIINc [ 54 ].…”

“…It has been shown that substitution of a C120 by a phenylalanine or serine in PKSIIINc results in synthesis of phloroglucinol instead of α-pyrone [ 50 ]. The active site is flanked by the acyl-binding tunnel similar to those observed in the structures of bacterial and fungal PKSIII [ 38 , 39 , 47 , 50 , 53 ]. We observed some differences with residues observed in the tunnel of PKSIIINc [ 47 ].…”