Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes
ABSTRACT The nickel-pincer nucleotide (NPN) cofactor is a modified pyridinium mononucleotide that tri-coordinates nickel and is crucial for the activity of certain racemases and epimerases. LarB, LarC, and LarE are responsible for NPN synthesis, with the cofactor subsequently installed into LarA hom...
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| Language: | English |
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American Society for Microbiology
2025-02-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.03404-24 |
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| author | Jorge L. Nevarez Aiko Turmo Santhosh Gatreddi Swati Gupta Jian Hu Robert P. Hausinger |
| author_facet | Jorge L. Nevarez Aiko Turmo Santhosh Gatreddi Swati Gupta Jian Hu Robert P. Hausinger |
| author_sort | Jorge L. Nevarez |
| collection | DOAJ |
| description | ABSTRACT The nickel-pincer nucleotide (NPN) cofactor is a modified pyridinium mononucleotide that tri-coordinates nickel and is crucial for the activity of certain racemases and epimerases. LarB, LarC, and LarE are responsible for NPN synthesis, with the cofactor subsequently installed into LarA homologs. Hurdles for investigating the functional properties of such proteins arise from the difficulty of obtaining the active, NPN cofactor-loaded enzymes and in assaying their diverse reactivities. Here, we show that when the Lactiplantibacillus plantarum lar genes are cloned into the Duet expression system and cultured in Escherichia coli, they confer lactate racemase activity to the cells. By replacing L. plantarum larA with related genes from other microorganisms, this system allows for the generation of active LarA homologs. Furthermore, the Duet system enables the functional testing of LarB, LarC, and LarE homologs from other microorganisms. In addition to applying the Duet expression system for synthesis of active, NPN cofactor-containing enzymes in E. coli, we demonstrate that circular dichroism spectroscopy provides a broadly applicable means of assaying these enzymes. By selecting a wavelength of high molar ellipticity and low absorbance for a given 2-hydroxy acid substrate enantiomer, the conversion of one enantiomer/epimer into the other can be monitored for LarA homologs without the need for any coupling enzymes or reagents. The methods discussed here further our abilities to investigate the unique activities of Lar proteins.IMPORTANCEEnzymes containing the nickel-pincer nucleotide (NPN) cofactor are prevalent in a wide range of microorganisms and catalyze various critical biochemical reactions, yet they remain underexplored due, in part, to limitations in current research methodologies. The two significant advancements described here, the heterologous production of active NPN-cofactor containing enzymes in Escherichia coli and the use of a circular dichroism-based assay to monitor enzyme activities, expand our capacity to analyze these enzymes. Such additional detailed characterization will deepen our understanding of the diverse chemistry catalyzed by the NPN cofactor and potentially uncover novel roles for this organometallic species in microbial metabolism. |
| format | Article |
| id | doaj-art-4323daffc8ab484f8391304b70481b12 |
| institution | Kabale University |
| issn | 2150-7511 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj-art-4323daffc8ab484f8391304b70481b122025-02-05T14:00:48ZengAmerican Society for MicrobiologymBio2150-75112025-02-0116210.1128/mbio.03404-24Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymesJorge L. Nevarez0Aiko Turmo1Santhosh Gatreddi2Swati Gupta3Jian Hu4Robert P. Hausinger5Department of Chemistry, Michigan State University, East Lansing, Michigan, USADepartment of Microbiology, Genetics, and Immunology, Michigan State University, East Lansing, Michigan, USADepartment of Microbiology, Genetics, and Immunology, Michigan State University, East Lansing, Michigan, USADepartment of Microbiology, Genetics, and Immunology, Michigan State University, East Lansing, Michigan, USADepartment of Chemistry, Michigan State University, East Lansing, Michigan, USADepartment of Microbiology, Genetics, and Immunology, Michigan State University, East Lansing, Michigan, USAABSTRACT The nickel-pincer nucleotide (NPN) cofactor is a modified pyridinium mononucleotide that tri-coordinates nickel and is crucial for the activity of certain racemases and epimerases. LarB, LarC, and LarE are responsible for NPN synthesis, with the cofactor subsequently installed into LarA homologs. Hurdles for investigating the functional properties of such proteins arise from the difficulty of obtaining the active, NPN cofactor-loaded enzymes and in assaying their diverse reactivities. Here, we show that when the Lactiplantibacillus plantarum lar genes are cloned into the Duet expression system and cultured in Escherichia coli, they confer lactate racemase activity to the cells. By replacing L. plantarum larA with related genes from other microorganisms, this system allows for the generation of active LarA homologs. Furthermore, the Duet system enables the functional testing of LarB, LarC, and LarE homologs from other microorganisms. In addition to applying the Duet expression system for synthesis of active, NPN cofactor-containing enzymes in E. coli, we demonstrate that circular dichroism spectroscopy provides a broadly applicable means of assaying these enzymes. By selecting a wavelength of high molar ellipticity and low absorbance for a given 2-hydroxy acid substrate enantiomer, the conversion of one enantiomer/epimer into the other can be monitored for LarA homologs without the need for any coupling enzymes or reagents. The methods discussed here further our abilities to investigate the unique activities of Lar proteins.IMPORTANCEEnzymes containing the nickel-pincer nucleotide (NPN) cofactor are prevalent in a wide range of microorganisms and catalyze various critical biochemical reactions, yet they remain underexplored due, in part, to limitations in current research methodologies. The two significant advancements described here, the heterologous production of active NPN-cofactor containing enzymes in Escherichia coli and the use of a circular dichroism-based assay to monitor enzyme activities, expand our capacity to analyze these enzymes. Such additional detailed characterization will deepen our understanding of the diverse chemistry catalyzed by the NPN cofactor and potentially uncover novel roles for this organometallic species in microbial metabolism.https://journals.asm.org/doi/10.1128/mbio.03404-24nickelcofactorracemaseepimerasecircular dichroism spectroscopy |
| spellingShingle | Jorge L. Nevarez Aiko Turmo Santhosh Gatreddi Swati Gupta Jian Hu Robert P. Hausinger Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes mBio nickel cofactor racemase epimerase circular dichroism spectroscopy |
| title | Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes |
| title_full | Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes |
| title_fullStr | Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes |
| title_full_unstemmed | Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes |
| title_short | Overcoming barriers for investigating nickel-pincer nucleotide cofactor-related enzymes |
| title_sort | overcoming barriers for investigating nickel pincer nucleotide cofactor related enzymes |
| topic | nickel cofactor racemase epimerase circular dichroism spectroscopy |
| url | https://journals.asm.org/doi/10.1128/mbio.03404-24 |
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