STRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis
Polyketides are secondary metabolites produced by plants, animals, fungi, and bacteria. Polyketide is synthesized through condensation reaction of C2 multiple carbon chain of fatty acid. α-Pyrone is an example of polyketide which is embedded in lipid and glycolipid layer of Mycobacterium tu...
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Polyketides are secondary metabolites produced by plants, animals, fungi, and bacteria. Polyketide is synthesized through condensation reaction of C2 multiple carbon chain of fatty acid. α-Pyrone is an example of polyketide which is embedded in lipid and glycolipid layer of Mycobacterium tuberculosis cell wall. Polyketide biosynthesis is catalysed by Polyketide synthase (PKS). Based on number of subunits and mode of synthesis, PKSs are divided into three types, namely type I PKS, type II PKS, and type III PKS. PKS18 of M. tuberculosis belongs to a type III PKS which catalyses the synthesis of α-pyrone. This research aimed to study structure and function of type III PKS from two clinical isolates M. tuberculosis. M. tuberculosis clinical isolates used in this study were M. tuberculosis L26 and H37. The PCR amplified pks18 gene M. tuberculosis was first cloned into the pGEM-T vector, subcloned into pET-30a(+) expression vector, and then expressed in Escherichia coli BL21 (DE3). The recombinant PKS18 was purified and characterized in term of its ability to <br />
<br />
catalyse the synthesis of α-pyrone polyketide. The pks18 gene was amplified using a set of primers which were designed based on M. tuberculosis strain H37Rv (GenBank accession no. P9WPFI) in which the sequence of forward primer was 5'-ggatccATGAACGTCTCAGCTGAGAG-3' and the reverse was 5'-aagcttTCACCGTCGGATGATGTCGA-3'. The amplicon <br />
<br />
with the size of 1.2 kb was cloned into the pGEM-T vector. This had been inserted into pET-30a(+) expression plasmid generating pET-pks18 recombinant plasmid. <br />
<br />
Nucleotide sequence alignment analysis of pks18 among M. tuberculosis strain H37Rv (as a reference strain) and the two M. tuberculosis clinical isolates demonstrated pks18 of L26 was identical with the pks18 of reference strain, while pks18 of H37 has two nucleotide changes. The pks18 of H37 has mutation of C823T and C1142T which results in amino acid substitutions of Cys275Arg and Val381Ala. These types of mutations have never been reported. The pks18 gene were expressed in E. coli strain BL21 (DE3) employing IPTG addition as an inducer. SDS-PAGE analysis showed that the PKS18 from L26 (PKS18_L26) and PKS18 from H37 (PKS18_H37) were both produced as soluble and inclusion bodies (aggregate) with molecular weight of ~48 kDa. <br />
<br />
Protein band intensity indicated that the amount inclusion bodies of PKS18_H37 was much higher compared to PKS18_L26 suggesting that the solubility of PKS18_H37 is less than that of PKS18_L26. The abilities of PKS18_L26 and PKS18_H37 in catalysing α-pyrone polyketide biosynthesis were studied using malonyl-CoA as an extender and hexanoyl-CoA as a starter. The expected products were analysed by TLC. TLC chromatogram indicated the product of reaction synthesized by PKS18_L26 was detected as new <br />
<br />
spot with Rf higher than both malonyl-CoA and hexanoyl-CoA, whereas no product appeared from reaction by PKS18_H37. Furthermore, action of PKS18 was also performed using malonyl-CoA as a starter and extender. The HPLC <br />
<br />
chromatogram of the product indicated that PKS18_L26 was able to catalyse the biosynthesis of α-pyrone polyketide. In contrast, PKS18_H37 has lost its ability to catalyze α-pyrone polyketide biosynthesis indicating that PKS18_H37 is inactive. Protein model structure analysis showed that PKS18_L26 has similar structure as PKS18_H37Rv. On the other hand, PKS18_H37 having substitution of Cys275Arg apparently has altered secondary structure, whereas substitution of Val381Ala in PKS18_37 gave no effect on structure. Since Cys275 is located in substrates binding tunnel, this alteration might cause constriction and in turn would inhibit substrates to reach the catalytic site. <br />
<br />
This research clearly indicated the correlation between PKS18 structure and PKS18 function in catalysing α-pyrone polyketide biosynthesis. Therefore, in the future it is important to perform a more thorough study on three dimensional structure of PKS18_H37, to isolate α-pyrone from the two M. tuberculosis clinical isolates to elucidate the function of PKS18 in vivo. The study of PKS18 from various different clinical isolates of M. tuberculosis would also be of interest in order to understand the correlation between the presence of α-pyron in cell wall and drug sensitivity. |
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Dissertations |
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BUDI SATIYARTI (NIM : 30510008), RINA |
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BUDI SATIYARTI (NIM : 30510008), RINA STRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis |
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BUDI SATIYARTI (NIM : 30510008), RINA |
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BUDI SATIYARTI (NIM : 30510008), RINA |
title |
STRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis |
title_short |
STRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis |
title_full |
STRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis |
title_fullStr |
STRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis |
title_full_unstemmed |
STRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis |
title_sort |
structure and function of polyketide synthase (pks18) from mycobacterium tuberculosis |
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id-itb.:239992017-11-22T15:24:43ZSTRUCTURE AND FUNCTION of POLYKETIDE SYNTHASE (PKS18) from Mycobacterium tuberculosis BUDI SATIYARTI (NIM : 30510008), RINA Indonesia Dissertations INSTITUT TEKNOLOGI BANDUNG https://digilib.itb.ac.id/gdl/view/23999 Polyketides are secondary metabolites produced by plants, animals, fungi, and bacteria. Polyketide is synthesized through condensation reaction of C2 multiple carbon chain of fatty acid. α-Pyrone is an example of polyketide which is embedded in lipid and glycolipid layer of Mycobacterium tuberculosis cell wall. Polyketide biosynthesis is catalysed by Polyketide synthase (PKS). Based on number of subunits and mode of synthesis, PKSs are divided into three types, namely type I PKS, type II PKS, and type III PKS. PKS18 of M. tuberculosis belongs to a type III PKS which catalyses the synthesis of α-pyrone. This research aimed to study structure and function of type III PKS from two clinical isolates M. tuberculosis. M. tuberculosis clinical isolates used in this study were M. tuberculosis L26 and H37. The PCR amplified pks18 gene M. tuberculosis was first cloned into the pGEM-T vector, subcloned into pET-30a(+) expression vector, and then expressed in Escherichia coli BL21 (DE3). The recombinant PKS18 was purified and characterized in term of its ability to <br /> <br /> catalyse the synthesis of α-pyrone polyketide. The pks18 gene was amplified using a set of primers which were designed based on M. tuberculosis strain H37Rv (GenBank accession no. P9WPFI) in which the sequence of forward primer was 5'-ggatccATGAACGTCTCAGCTGAGAG-3' and the reverse was 5'-aagcttTCACCGTCGGATGATGTCGA-3'. The amplicon <br /> <br /> with the size of 1.2 kb was cloned into the pGEM-T vector. This had been inserted into pET-30a(+) expression plasmid generating pET-pks18 recombinant plasmid. <br /> <br /> Nucleotide sequence alignment analysis of pks18 among M. tuberculosis strain H37Rv (as a reference strain) and the two M. tuberculosis clinical isolates demonstrated pks18 of L26 was identical with the pks18 of reference strain, while pks18 of H37 has two nucleotide changes. The pks18 of H37 has mutation of C823T and C1142T which results in amino acid substitutions of Cys275Arg and Val381Ala. These types of mutations have never been reported. The pks18 gene were expressed in E. coli strain BL21 (DE3) employing IPTG addition as an inducer. SDS-PAGE analysis showed that the PKS18 from L26 (PKS18_L26) and PKS18 from H37 (PKS18_H37) were both produced as soluble and inclusion bodies (aggregate) with molecular weight of ~48 kDa. <br /> <br /> Protein band intensity indicated that the amount inclusion bodies of PKS18_H37 was much higher compared to PKS18_L26 suggesting that the solubility of PKS18_H37 is less than that of PKS18_L26. The abilities of PKS18_L26 and PKS18_H37 in catalysing α-pyrone polyketide biosynthesis were studied using malonyl-CoA as an extender and hexanoyl-CoA as a starter. The expected products were analysed by TLC. TLC chromatogram indicated the product of reaction synthesized by PKS18_L26 was detected as new <br /> <br /> spot with Rf higher than both malonyl-CoA and hexanoyl-CoA, whereas no product appeared from reaction by PKS18_H37. Furthermore, action of PKS18 was also performed using malonyl-CoA as a starter and extender. The HPLC <br /> <br /> chromatogram of the product indicated that PKS18_L26 was able to catalyse the biosynthesis of α-pyrone polyketide. In contrast, PKS18_H37 has lost its ability to catalyze α-pyrone polyketide biosynthesis indicating that PKS18_H37 is inactive. Protein model structure analysis showed that PKS18_L26 has similar structure as PKS18_H37Rv. On the other hand, PKS18_H37 having substitution of Cys275Arg apparently has altered secondary structure, whereas substitution of Val381Ala in PKS18_37 gave no effect on structure. Since Cys275 is located in substrates binding tunnel, this alteration might cause constriction and in turn would inhibit substrates to reach the catalytic site. <br /> <br /> This research clearly indicated the correlation between PKS18 structure and PKS18 function in catalysing α-pyrone polyketide biosynthesis. Therefore, in the future it is important to perform a more thorough study on three dimensional structure of PKS18_H37, to isolate α-pyrone from the two M. tuberculosis clinical isolates to elucidate the function of PKS18 in vivo. The study of PKS18 from various different clinical isolates of M. tuberculosis would also be of interest in order to understand the correlation between the presence of α-pyron in cell wall and drug sensitivity. text |