Cloning, expression, purification, and characterization of glutamate decarboxylase (Rv3432c) from Mycobacterium tuberculosis.
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| Title: | Cloning, expression, purification, and characterization of glutamate decarboxylase (Rv3432c) from Mycobacterium tuberculosis. |
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| Authors: | Rai, Rupal1 (AUTHOR), Paroha, Ruchi2 (AUTHOR), Rai, Sandesh3 (AUTHOR), Singh, Anirudh K.4 (AUTHOR), Chaurasia, Rashmi5 (AUTHOR), Agarwal, Nisheeth6 (AUTHOR), Pandey, Megha Katare7 (AUTHOR), Chaurasiya, Shivendra K.1 (AUTHOR) shivendrachaurasiya@gmail.com |
| Source: | International Microbiology. Oct2025, Vol. 28 Issue 7, p1603-1616. 14p. |
| Abstract: | Glutamate decarboxylase (Gad), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA), consuming a proton in the process and thereby contributing to intracellular pH homeostasis in bacteria. However, the presence and function of the Gad-dependent mechanism in mycobacteria remain largely unexplored. This study aimed to characterize Gad activity in Mycobacterium tuberculosis (Mtb). We detected Gad activity in live cells of both Mtb and Mycobacterium smegmatis (MS). Gad activity and GABA was also detected in cell lysates of Mtb and MS. The gadB gene from Mtb was cloned, expressed, and GadB protein was purified under native conditions using MS as an expression host. Initial attempts to express GadB in Escherichia coli (E. coli) resulted in the overexpressed protein being present in the insoluble fraction and was enzymatically inactive when purified under denaturing conditions. Subsequently, an acetamide-inducible expression system was employed in MS for successful overexpression and purification of recombinant GadB. 6 × His-GadB was purified using immobilized metal affinity chromatography, and its molecular weight was determined to be ~ 51.2 kDa by SDS-PAGE. The purified 6 × His-GadB enzyme was active at both neutral and acidic pH. Its activity was found to be PLP-dependent, with optimal activity at pH 7.2 and 50°C. These findings suggest that Gad is expressed in Mtb both in normal and in acidic medium, supporting the possible existence of a Gad-dependent acid resistance mechanism in Mtb. [ABSTRACT FROM AUTHOR] |
| Copyright of International Microbiology is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
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| Header | DbId: egs DbLabel: Engineering Source An: 188686188 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: Cloning, expression, purification, and characterization of glutamate decarboxylase (Rv3432c) from Mycobacterium tuberculosis. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Rai%2C+Rupal%22">Rai, Rupal</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Paroha%2C+Ruchi%22">Paroha, Ruchi</searchLink><relatesTo>2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Rai%2C+Sandesh%22">Rai, Sandesh</searchLink><relatesTo>3</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Singh%2C+Anirudh+K%2E%22">Singh, Anirudh K.</searchLink><relatesTo>4</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Chaurasia%2C+Rashmi%22">Chaurasia, Rashmi</searchLink><relatesTo>5</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Agarwal%2C+Nisheeth%22">Agarwal, Nisheeth</searchLink><relatesTo>6</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Pandey%2C+Megha+Katare%22">Pandey, Megha Katare</searchLink><relatesTo>7</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Chaurasiya%2C+Shivendra+K%2E%22">Chaurasiya, Shivendra K.</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> shivendrachaurasiya@gmail.com</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22International+Microbiology%22">International Microbiology</searchLink>. Oct2025, Vol. 28 Issue 7, p1603-1616. 14p. – Name: Abstract Label: Abstract Group: Ab Data: Glutamate decarboxylase (Gad), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA), consuming a proton in the process and thereby contributing to intracellular pH homeostasis in bacteria. However, the presence and function of the Gad-dependent mechanism in mycobacteria remain largely unexplored. This study aimed to characterize Gad activity in Mycobacterium tuberculosis (Mtb). We detected Gad activity in live cells of both Mtb and Mycobacterium smegmatis (MS). Gad activity and GABA was also detected in cell lysates of Mtb and MS. The gadB gene from Mtb was cloned, expressed, and GadB protein was purified under native conditions using MS as an expression host. Initial attempts to express GadB in Escherichia coli (E. coli) resulted in the overexpressed protein being present in the insoluble fraction and was enzymatically inactive when purified under denaturing conditions. Subsequently, an acetamide-inducible expression system was employed in MS for successful overexpression and purification of recombinant GadB. 6 × His-GadB was purified using immobilized metal affinity chromatography, and its molecular weight was determined to be ~ 51.2 kDa by SDS-PAGE. The purified 6 × His-GadB enzyme was active at both neutral and acidic pH. Its activity was found to be PLP-dependent, with optimal activity at pH 7.2 and 50°C. These findings suggest that Gad is expressed in Mtb both in normal and in acidic medium, supporting the possible existence of a Gad-dependent acid resistance mechanism in Mtb. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of International Microbiology is the property of Springer Nature and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1007/s10123-025-00637-8 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 14 StartPage: 1603 Titles: – TitleFull: Cloning, expression, purification, and characterization of glutamate decarboxylase (Rv3432c) from Mycobacterium tuberculosis. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Rai, Rupal – PersonEntity: Name: NameFull: Paroha, Ruchi – PersonEntity: Name: NameFull: Rai, Sandesh – PersonEntity: Name: NameFull: Singh, Anirudh K. – PersonEntity: Name: NameFull: Chaurasia, Rashmi – PersonEntity: Name: NameFull: Agarwal, Nisheeth – PersonEntity: Name: NameFull: Pandey, Megha Katare – PersonEntity: Name: NameFull: Chaurasiya, Shivendra K. IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 10 Text: Oct2025 Type: published Y: 2025 Identifiers: – Type: issn-print Value: 11396709 Numbering: – Type: volume Value: 28 – Type: issue Value: 7 Titles: – TitleFull: International Microbiology Type: main |
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