Expression and kinetic characterization of PYCR3

Meeks, Kaylen R.; Tanner, John J.

doi:10.1016/j.abb.2022.109468

Cited by 3 publications

(2 citation statements)

References 20 publications

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“…Recombinant PYCR3 was expressed as a SUMO fusion and purified as described previously (Meeks & Tanner, 2023). PYCR3 activity was assayed with the same experimental setup as PYCR1.…”

Section: Methodsmentioning

confidence: 99%

Screening a knowledge‐based library of low molecular weight compounds against the proline biosynthetic enzyme 1‐pyrroline‐5‐carboxylate 1 (PYCR1)

Meeks,

Bogner,

Tanner

2024

Protein Science

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AbstractΔ1‐pyrroline‐5‐carboxylate reductase isoform 1 (PYCR1) is the last enzyme of proline biosynthesis and catalyzes the NAD(P)H‐dependent reduction of Δ1‐pyrroline‐5‐carboxylate to L‐proline. High PYCR1 gene expression is observed in many cancers and linked to poor patient outcomes and tumor aggressiveness. The knockdown of the PYCR1 gene or the inhibition of PYCR1 enzyme has been shown to inhibit tumorigenesis in cancer cells and animal models of cancer, motivating inhibitor discovery. We screened a library of 71 low molecular weight compounds (average MW of 131 Da) against PYCR1 using an enzyme activity assay. Hit compounds were validated with X‐ray crystallography and kinetic assays to determine affinity parameters. The library was counter‐screened against human Δ1‐pyrroline‐5‐carboxylate reductase isoform 3 and proline dehydrogenase (PRODH) to assess specificity/promiscuity. Twelve PYCR1 and one PRODH inhibitor crystal structures were determined. Three compounds inhibit PYCR1 with competitive inhibition parameter of 100 μM or lower. Among these, (S)‐tetrahydro‐2H‐pyran‐2‐carboxylic acid (70 μM) has higher affinity than the current best tool compound N‐formyl‐l‐proline, is 30 times more specific for PYCR1 over human Δ1‐pyrroline‐5‐carboxylate reductase isoform 3, and negligibly inhibits PRODH. Structure‐affinity relationships suggest that hydrogen bonding of the heteroatom of this compound is important for binding to PYCR1. The structures of PYCR1 and PRODH complexed with 1‐hydroxyethane‐1‐sulfonate demonstrate that the sulfonate group is a suitable replacement for the carboxylate anchor. This result suggests that the exploration of carboxylic acid isosteres may be a promising strategy for discovering new classes of PYCR1 and PRODH inhibitors. The structure of PYCR1 complexed with l‐pipecolate and NADH supports the hypothesis that PYCR1 has an alternative function in lysine metabolism.

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“…Recombinant PYCR3 was expressed as a SUMO fusion and purified as described previously (Meeks & Tanner, 2023). PYCR3 activity was assayed with the same experimental setup as PYCR1.…”

Section: Methodsmentioning

confidence: 99%

Screening a knowledge‐based library of low molecular weight compounds against the proline biosynthetic enzyme 1‐pyrroline‐5‐carboxylate 1 (PYCR1)

Meeks,

Bogner,

Tanner

2024

Protein Science

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“…NADPH (reduced nicotinamide adenine dinucleotide phosphate) is a central cofactor in a wide range of biosynthetic pathways that are essential in the metabolism of plant cells, such as the Calvin cycle, carotenoids, fatty acids and the aromatic amino acids (Phe, Tyr, Trp) as well as proline [ 1 , 2 , 3 ]. Furthermore, NADPH also sustains cellular detoxification and defense; for example, it is necessary to support glutathione reductase activity as part of the ascorbate–glutathione cycle and the generation of superoxide radicals by NADPH oxidase (NOX), which is also designated as the respiratory burst oxidase homolog (Rboh) [ 4 , 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

NADP-Dependent Malic Enzyme Genes in Sweet Pepper Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO)

Taboada

González‐Gordo

Muñoz‐Vargas

et al. 2023

Plants

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NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50–75% ammonium–sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment.

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PYCR3 modulates mtDNA copy number to drive proliferation and doxorubicin resistance in triple-negative breast cancer

Zhuang,

Huang,

Liu

2024

The International Journal of Biochemistry & Cell Biology

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Expression and kinetic characterization of PYCR3

Cited by 3 publications

References 20 publications

Screening a knowledge‐based library of low molecular weight compounds against the proline biosynthetic enzyme 1‐pyrroline‐5‐carboxylate 1 (PYCR1)

Screening a knowledge‐based library of low molecular weight compounds against the proline biosynthetic enzyme 1‐pyrroline‐5‐carboxylate 1 (PYCR1)

NADP-Dependent Malic Enzyme Genes in Sweet Pepper Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO)

PYCR3 modulates mtDNA copy number to drive proliferation and doxorubicin resistance in triple-negative breast cancer

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