Crystal structure of dihydrodipicolinate reductase (PaDHDPR) from Paenisporosarcina sp. TG-14: structural basis for NADPH preference as a cofactor

Lee, Chang Woo; Park, Sunha; Lee, Sung Gu; Park, Hyun Ho; Kim, Soo Hyun; Park, HaJeung; Park, Hyun; Lee, Jun Hyuck

doi:10.1038/s41598-018-26291-x

Cited by 9 publications

(3 citation statements)

References 52 publications

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“…Multisubstrate enzyme mechanisms are difficult to study with conventional techniques. Elucidating such mechanisms requires the determination of the binding order of the substrates − and the determination of inhibition modes by the substrate. Therefore, the use of ITC seemed worthwhile to gain mechanistic insights and to elicit the robustness of our approach.…”

Section: Resultsmentioning

confidence: 99%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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To apply enzymes in technical processes, a detailed understanding of the molecular mechanisms is required. Kinetic and thermodynamic parameters of enzyme catalysis are crucial to plan, model, and implement biocatalytic processes more efficiently. While the kinetic parameters, K m and k cat , are often accessible by optical methods, the determination of thermodynamic parameters requires more sophisticated methods. Isothermal titration calorimetry (ITC) allows the label-free and highly sensitive analysis of kinetic and thermodynamic parameters of individual steps in the catalytic cycle of an enzyme reaction. However, since ITC is susceptible to interferences due to denaturation or agglomeration of the enzymes, the homogeneity of the enzyme sample must always be considered, and this can be accomplished by means of dynamic light scattering (DLS) analysis. We here report on the use of an ITC-dependent work flow to determine both the kinetic and the thermodynamic data for a cofactor-dependent enzyme. Using a standardized approach with the implementation of sample quality control by DLS, we obtain high-quality data suitable for the advanced modeling of the enzyme reaction mechanism. Specifically, we investigated stereoselective reactions catalyzed by the NADPH-dependent ketoreductase Gre2p under different reaction conditions. The results revealed that this enzyme operates with an ordered sequential mechanism and is affected by substrate or product inhibition depending on the reaction buffer. Data reproducibility is ensured by specifying standard operating procedures, using programmed workflows for data analysis, and storing all data in a F.A.I.R. (findable, accessible, interoperable, and reusable) repository (https://doi.org/10.15490/fairdomhub.1.investigation.464.1). Our work highlights the utility for combined binding and kinetic studies for such complex multisubstrate reactions.

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Section: Resultsmentioning

confidence: 99%

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

et al. 2021

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“…The DapF1 (Zm00001d030677) identi ed in our study in QTL qLYS-4-1 might be an important gene involved in lysine biosynthesis. Dihydrodipicolinate reductase (DapB) catalyses the second reaction in the diaminopimelate pathway of lysine biosynthesis in the diaminopimelate pathway [6,[48][49][50][51]. In this study, we identi ed two DapB genes, DapB1 (Zm00001d047935) and DapB2 (Zm00001d049956) on chromosome 9 and 4, respectively, which might be responsible for the key enzymes in the diaminopimelate-and lysine-synthesis pathways that reduces dihydrodipicolinate to tetrahydrodipicolinate.…”

Section: Genetic Basis Of Lysine Content In Dh Populationsmentioning

confidence: 99%

Genetic analysis of QTLs for lysine content in four maize DH populations

Zhang,

Wen,

Wang

et al. 2024

Preprint

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Background Low level of lysine in maize endosperm is considered to be a major problem for determining the nutritional quality of food and feed. Improving the lysine content is favorable to improve maize quality by optimizing feeding requirement. Understanding the genetic basis of lysine content benefits greatly improving maize yield and optimizing end-use quality. Results Four double haploid (DH) populations were generated and used to identify quantitative trait loci (QTL) associated with lysine content. The broad-sense heritability indicated the majority of lysine content variations were largely controlled by genetic factors. A total of 12 QTLs were identified in a range of 4.42–12.66% in term of phenotypic variation explained (PVE) which suggested that a large number of minor-effect QTLs mainly contributed to the genetic component of lysine content. Five well-known genes encoding key enzymes in maize lysine biosynthesis pathways locate within QTLs identified in this study. Conclusions The information presented will pave a path to explore candidate genes regulating lysine biosynthesis pathways and be useful for marker-assisted selection and gene pyramiding in high-lysine maize breeding programs.

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“…Multi-reactant enzyme mechanisms are difficult to study with conventional techniques. Elucidating such mechanisms requires the determination of the binding order of the reactants, [34][35][36] and the determination of inhibition modes by the reactants. Therefore, the use of ITC seemed worthwhile to gain mechanistic insights and to elicit the robustness of our approach.…”

Section: Introducing Gre2pmentioning

confidence: 99%

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Ott¹,

Rabe²,

Niemeyer³

et al. 2021

Preprint

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<div> <p>An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are dynamic light scattering (DLS), UV-Vis spectrophotometry and isothermal titration calorimetry (ITC) respectively. The workflow is standardized by the use of SOPs and python-scripted data analysis. </p> <p>We have used the NADPH-dependent alcohol dehydrogenase Gre2p as a challenging enzyme to demonstrate the power of this workflow. Our work highlights the utility for combined binding and kinetic studies for such complex multi-substrate reactions and the importance of sample quality control during experiments.</p> </div>

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Crystal structure of dihydrodipicolinate reductase (PaDHDPR) from Paenisporosarcina sp. TG-14: structural basis for NADPH preference as a cofactor

Cited by 9 publications

References 52 publications

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Toward Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

Genetic analysis of QTLs for lysine content in four maize DH populations

Towards Reproducible Enzyme Modeling with Isothermal Titration Calorimetry

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