Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi

Dhivya Ramakrishnan; Manish Kumar Tiwari; Gomathi Manoharan; Thiagarajan Sairam; Rajesh Thangamani; Jung Kul Lee; Jeya Marimuthu

doi:10.1016/j.enzmictec.2018.04.006

Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi

Dhivya Ramakrishnan, Manish Kumar Tiwari, Gomathi Manoharan, Thiagarajan Sairam, Rajesh Thangamani, Jung Kul Lee, Jeya Marimuthu^*

^*Corresponding author af dette arbejde

Kemisk Institut

4 Citationer (Scopus)

Abstract

Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C₄–C₂₀) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 10⁴ s⁻¹ M⁻¹ catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 10⁴ s⁻¹ M⁻¹. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.

Originalsprog	Engelsk
Tidsskrift	Enzyme and Microbial Technology
Vol/bind	115
Sider (fra-til)	16-22
Antal sider	7
ISSN	0141-0229
DOI	https://doi.org/10.1016/j.enzmictec.2018.04.006
Status	Udgivet - 2018

Adgang til dokumentet

10.1016/j.enzmictec.2018.04.006

Citationsformater

@article{0898e124adb4458da775a84c05fa1e38,

title = "Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi",

abstract = "Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4–C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s−1 M−1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s−1 M−1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.",

keywords = "Catalytic efficiency, Chaetomium thermophilum, Resorcylic acid, Sordaria macrospora, Substrate specificity, Type III polyketide synthase",

author = "Dhivya Ramakrishnan and Tiwari, {Manish Kumar} and Gomathi Manoharan and Thiagarajan Sairam and Rajesh Thangamani and Lee, {Jung Kul} and Jeya Marimuthu",

year = "2018",

doi = "10.1016/j.enzmictec.2018.04.006",

language = "English",

volume = "115",

pages = "16--22",

journal = "Enzyme and Microbial Technology",

issn = "0141-0229",

publisher = "Elsevier",

}

TY - JOUR

T1 - Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi

AU - Ramakrishnan, Dhivya

AU - Tiwari, Manish Kumar

AU - Manoharan, Gomathi

AU - Sairam, Thiagarajan

AU - Thangamani, Rajesh

AU - Lee, Jung Kul

AU - Marimuthu, Jeya

PY - 2018

Y1 - 2018

N2 - Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4–C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s−1 M−1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s−1 M−1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.

AB - Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4–C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s−1 M−1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s−1 M−1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.

KW - Catalytic efficiency

KW - Chaetomium thermophilum

KW - Resorcylic acid

KW - Sordaria macrospora

KW - Substrate specificity

KW - Type III polyketide synthase

U2 - 10.1016/j.enzmictec.2018.04.006

DO - 10.1016/j.enzmictec.2018.04.006

M3 - Journal article

C2 - 29859598

AN - SCOPUS:85045579922

SN - 0141-0229

VL - 115

SP - 16

EP - 22

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

ER -

Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi

Abstract

Adgang til dokumentet

Fingeraftryk

Citationsformater