The NADP+-dependent glutamate dehydrogenase of the yeast Kluyveromyces marxianus responds to nitrogen repression similarly to Saccharomyces cerevisiae

MoraisJunior, Marcos Antonio de

doi:10.1590/s1517-83822003000400009

Cited by 8 publications

(3 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The unique characteristics of the obtained GDH with optimum pH at 4.0 and NAD inhibition distinguish it from other GDHs that were not been previously reported to have activity towards higher alcohols. The reported NADP + -dependent GDH from Kluyveromyces marxianus showed a significantly lower specific growth rate on ethanol, and its activity was identical with the presence of glucose (Morais-Júnior 2003). Furthermore, the obtained enzyme was also different from all reported NAD- and NADP-dependent GDHs that occupy a key position in inter-linking carbon and nitrogen metabolism in fungi.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity

Zhu

et al. 2017

AMB Expr

View full text Add to dashboard Cite

Crude enzyme from Geotrichum candidum S12 exhibited high activity towards hexanol at pH 4.0, distinguishing it from currently known enzymes. To identify the dominant enzyme contributing to this activity, the crude enzyme extract was separated into different fractions by ammonium sulfate precipitation, MonoQ anion-exchange chromatography, and Sephacryl S-200 gel filtration chromatography. Afraction with high activity towards hexanol at pH 4.0 was obtained, exhibiting 38-fold improved purity and a specific activity of 3802.7 U/mg. After electrophoretic analysis, the fraction showed a molecular weight of 223 kDa by Native-PAGE and 51.4 kDa by SDS-PAGE. The purified fraction was identified as a glutamate dehydrogenase (GDH) by peptide mass fingerprinting data. This fraction showed high activity towards glutamate, α-ketoglutarate, hexanol, and isoamyl alcohol with a Km value of 41.74, 4.01, 20.37, and 19.37 mM, respectively, but with no activity towards methanol, ethanol, 1-propanol, and isobutanol. As a comparison, the GDH from yeast had no activity towards hexanol and other alcohols. Kinetic analysis revealed that glutamate and hexanol served as competitive inhibitors to each other for the purified GDH. The GDH showed the highest activity towards hexanol at pH 4.0 and 30 °C, and was the most stable at pH 2.2–7.0 and ≤40 °C. The presence of ADP, Fe2+, K+, and Zn2+ increased the enzymatic activity towards hexanol and EDTA, Pb2+, Mn2+, ATP, and DTT decreased the activity. These novel characteristics expand the reported properties of GDH and suggest the newly characterized GDH has unique potential for practical application.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0307-8) contains supplementary material, which is available to authorized users.

show abstract

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity

Zhu

et al. 2017

AMB Expr

View full text Add to dashboard Cite

show abstract

“…The PCR product was first cloned into a pMD18-T Simple Vector, restriction-digested with BamHI and HindIII to obtain the gene insert, and then ligated into BamHI/HindIII-digested pET28as. The pET28as-GDH was sequenced, transformed into Escherichia coli BL21 (DE3), and cultured for GDH expression [21].…”

Section: Expression and Purification Of Gcgdhmentioning

confidence: 99%

Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from Geotrichum candidum

Zhu

Hou

et al. 2023

Crystals

View full text Add to dashboard Cite

To better understand its mechanism of activity towards higher alcohols, we overexpressed and purified new Geotrichum candidum GDH (GcGDH). The purified GcGDH (50.27 kDa) was then crystallized, and the crystal diffracted to a resolution of 2.3 Å using X-ray diffraction. We found that the GcGDH crystal structure belonged to space group P212121 and was comprised of two hexamers organized into an asymmetric unit, with each subunit consisting of 452 amino acid residues. The binding sites between higher alcohols or L-glutamic acid and GcGDH were consistent. The optimal reaction conditions for GcGDH and hexanol were a pH of 4.0 and temperature of 30 °C, and those for GcGDH and monosodium glutamate (MSG) were a pH of 8.0 and temperature of 20 °C. The Km values for hexanol and MSG were found to be 74.78 mM and 0.018 mM, respectively. Mutating GcGDH Lys 113 to either Ala or Gly caused a dramatic reduction in its catalytic efficiency towards both MSG and hexanol, suggesting that Lys 113 is essential to the active site of GcGDH.

show abstract

“…cerevisiae, glutamate synthesis via NADPH dependent glutamate dehydrogenase is subject to nitrogen catabolite repression [59]. To analyse these type of product -substrate interactions, kinetic modelling would need to be applied.…”

Section: Glutamate Productionmentioning

confidence: 99%

Model-based biotechnological potential analysis of Kluyveromyces marxianus central metabolism

Pentjuss

Stalidzāns

Liepiņš

et al. 2017

Journal of Industrial Microbiology and Biotechnology

View full text Add to dashboard Cite

The non-conventional yeast Kluyveromyces marxianus is an emerging industrial producer for many biotechnological processes. Here, we show the application of a biomass-linked stoichiometric model of central metabolism that is experimentally validated, and mass and charge balanced for assessing the carbon conversion efficiency of wild type and modified K. marxianus. Pairs of substrates (lactose, glucose, inulin, xylose) and products (ethanol, acetate, lactate, glycerol, ethyl acetate, succinate, glutamate, phenylethanol and phenylalanine) are examined by various modelling and optimisation methods. Our model reveals the organism's potential for industrial application and metabolic engineering. Modelling results imply that the aeration regime can be used as a tool to optimise product yield and flux distribution in K. marxianus. Also rebalancing NADH and NADPH utilisation can be used to improve the efficiency of substrate conversion. Xylose is identified as a biotechnologically promising substrate for K. marxianus.

show abstract

The NADP+-dependent glutamate dehydrogenase of the yeast Kluyveromyces marxianus responds to nitrogen repression similarly to Saccharomyces cerevisiae

Cited by 8 publications

References 16 publications

Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity

Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity

Crystal Structure Analysis and Characterization of NADP-Dependent Glutamate Dehydrogenase with Alcohols Activity from Geotrichum candidum

Model-based biotechnological potential analysis of Kluyveromyces marxianus central metabolism

Contact Info

Product

Resources

About