Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall

Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified a...

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Main Authors: Neha eMalhotra, Pradip K Chakraborti
Format: Article
Language:English
Published: Frontiers Media S.A. 2016-02-01
Series:Frontiers in Microbiology
Subjects:
Online Access:http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.00123/full
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author Neha eMalhotra
Pradip K Chakraborti
author_facet Neha eMalhotra
Pradip K Chakraborti
author_sort Neha eMalhotra
collection DOAJ
description Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE) utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB and PknL), Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody marginally recognized mPDE-T309A mutant protein; however, such alteration did not affect the enzymatic activity. Interestingly, mPDE expressed in Mycobacterium smegmatis yielded a phosphorylated protein that preferentially localized to cell wall. In contrast, mPDE-T309A, the phosphoablative variant of mPDE, did not show such behaviour. On the other hand, phosphomimics of mPDE (T309D or T309E), exhibited similar cell wall anchorage as was observed with the wild-type. Thus, our results provide credence to the fact that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mPDE renders negative charge to the protein, promoting its localization on cell wall. Furthermore, multiple sequence alignment revealed that Thr-309 is conserved among mPDE orthologs of M. tuberculosis complex, which presumably emphasizes evolutionary significance of phosphorylation at this residue.
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spelling doaj.art-5817678919d4454a9fae0bbd46d60f442022-12-22T00:31:33ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2016-02-01710.3389/fmicb.2016.00123181092Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wallNeha eMalhotra0Pradip K Chakraborti1CSIR-Institute of Microbial TechnologyCSIR-Institute of Microbial TechnologyPhosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE) utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB and PknL), Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody marginally recognized mPDE-T309A mutant protein; however, such alteration did not affect the enzymatic activity. Interestingly, mPDE expressed in Mycobacterium smegmatis yielded a phosphorylated protein that preferentially localized to cell wall. In contrast, mPDE-T309A, the phosphoablative variant of mPDE, did not show such behaviour. On the other hand, phosphomimics of mPDE (T309D or T309E), exhibited similar cell wall anchorage as was observed with the wild-type. Thus, our results provide credence to the fact that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mPDE renders negative charge to the protein, promoting its localization on cell wall. Furthermore, multiple sequence alignment revealed that Thr-309 is conserved among mPDE orthologs of M. tuberculosis complex, which presumably emphasizes evolutionary significance of phosphorylation at this residue.http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.00123/fullMutagenesisSignal TransductioncAMPintracellular traffickingphosphodiesteraseenzyme activity
spellingShingle Neha eMalhotra
Pradip K Chakraborti
Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall
Frontiers in Microbiology
Mutagenesis
Signal Transduction
cAMP
intracellular trafficking
phosphodiesterase
enzyme activity
title Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall
title_full Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall
title_fullStr Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall
title_full_unstemmed Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall
title_short Eukaryotic-type Ser/Thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall
title_sort eukaryotic type ser thr protein kinase mediated phosphorylation of mycobacterial phosphodiesterase affects its localization to the cell wall
topic Mutagenesis
Signal Transduction
cAMP
intracellular trafficking
phosphodiesterase
enzyme activity
url http://journal.frontiersin.org/Journal/10.3389/fmicb.2016.00123/full
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