scholarly journals Metabolic Function of Corynebacterium glutamicum Aminotransferases AlaT and AvtA and Impact on l-Valine Production

2008 ◽  
Vol 74 (24) ◽  
pp. 7457-7462 ◽  
Author(s):  
Jan Marienhagen ◽  
Lothar Eggeling
Keyword(s):  
Corynebacterium Glutamicum ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Exponential Growth Phase ◽  
Dependent Manner ◽  
Wild Type ◽  
Growth Deficiency ◽  
In The Wild ◽  
Order Of Magnitude ◽  
Combined Data

ABSTRACT Aminotransferases (ATs) interacting with l-alanine are the least studied bacterial ATs. Whereas AlaT converts pyruvate to l-alanine in a glutamate-dependent reaction, AvtA is able to convert pyruvate to l-alanine in an l-valine-dependent manner. We show here that the wild type of Corynebacterium glutamicum with a deletion of either of the corresponding genes does not exhibit an explicit growth deficiency. However, a double mutant was auxotrophic for l-alanine, showing that both ATs can provide l-alanine and that they are the only ATs involved. Kinetic studies with isolated enzymes demonstrate that the catalytic efficiency, k cat/K m , of AlaT is higher than 1 order of magnitude in the direction of l-alanine formation (3.5 × 104 M−1 s−1), but no preference was apparent for AvtA, suggesting that AlaT is the principal l-alanine-supplying enzyme. This is in line with the cytosolic l-alanine concentration, which is reduced in the exponential growth phase from 95 mM to 18 mM by a deletion of alaT, whereas avtA deletion decreases the l-alanine concentration only to 76 mM. The combined data show that the presence of both ATs has subtle but obvious consequences on balancing intracellular amino acid pools in the wild type. The consequences are more obvious in an l-valine production strain where a high intracellular drain-off of the l-alanine precursor pyruvate prevails. We therefore used deletion of alaT to successfully reduce the contaminating l-alanine in extracellular accumulated l-valine by 80%.

scholarly journals Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

2008 ◽  
Vol 190 (19) ◽  
pp. 6458-6466 ◽  
Author(s):  
Jung-Won Youn ◽  
Elena Jolkver ◽  
Reinhard Krämer ◽  
Kay Marin ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Uptake System ◽  
Dependent Manner ◽  
Wild Type ◽  
Prolonged Incubation ◽  
In The Wild ◽  
Biochemical Analyses

ABSTRACT Many bacteria can utilize C4-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l-malate, and likely also oxaloacetate in a sodium-dependent manner.

scholarly journals Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

2003 ◽  
Vol 69 (5) ◽  
pp. 2521-2532 ◽  
Author(s):  
C. Lange ◽  
D. Rittmann ◽  
V. F. Wendisch ◽  
M. Bott ◽  
H. Sahm
Keyword(s):  
Corynebacterium Glutamicum ◽  
Expression Profiling ◽  
Large Subunit ◽  
Mrna Level ◽  
Acetohydroxyacid Synthase ◽  
Limiting Factor ◽  
Unexpected Result ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner

ABSTRACT Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

scholarly journals A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct

1997 ◽  
Vol 326 (3) ◽  
pp. 861-866 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
Regina M. DAY ◽  
Ekaterina V. TORCHILIN ◽  
William W. BACHOVCHIN ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Chemical Shifts ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Imidazolium Cation ◽  
Nmr Study ◽  
In The Wild ◽  
Main Factor

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3–103.6 p.p.m., depending on the pH. The titration shift of 4.7–4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.

scholarly journals Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

1994 ◽  
Vol 302 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A Schejter ◽  
T I Koshy ◽  
T L Luntz ◽  
R Sanishvili ◽  
I Vig ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
General Increase ◽  
Reduction Potentials ◽  
Cytochromes C ◽  
In The Wild ◽  
Order Of Magnitude ◽  
The Stability ◽  
Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

scholarly journals Computational modeling for antiarrhythmic drugs for atrial fibrillation according to the genotypes

EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
I Hwang ◽  
J Park ◽  
O Kwon ◽  
B Lim ◽  
M Hong ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Computational Modeling ◽  
Antiarrhythmic Drugs ◽  
Science Research ◽  
Dominant Frequency ◽  
Dependent Manner ◽  
Wild Type ◽  
National Budget ◽  
In The Wild ◽  
Public Grant

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by a grant [HI19C0114] from the Ministry of Health and Welfare. Additionally, the work was funded by grants [NRF-2019R1C1C100907512], and [NRF-2020R1A2B01001695] from the Basic Science Research Program run by the National Research Foundation of Korea (NRF) under the Ministry of Science, ICT & Future Planning (MSIP). Background The efficacy of antiarrhythmic drugs (AAD) can vary in patients with atrial fibrillation (AF) and the PITX2 gene affects the responsiveness of AADs. We explored the virtual AAD (V-AAD) responses between wild-type and PITX2+/- deficient AF conditions by realistic in-silico AF modeling. Methods We tested the V-AADs in AF modeling integrated with patients’ 3D-computed tomography and 3D-electroanatomical mapping, acquired in 25 patients (68% male, 59.8 ± 9.8 years old, 32.0% paroxysmal type). The ion currents for the PITX2+/- deficiency and each AAD (amiodarone, sotalol, dronedarone, flecainide, and propafenone) were defined based on previous publications. Results We compared the wild-type and PITX2+/- deficiency in terms of the action potential duration (APD90), conduction velocity (CV), maximal slope of restitution (Smax), and wave-dynamic parameters, such as the dominant frequency (DF), phase singularities (PS), and AF termination rates according to the V-AADs. The PITX2+/- deficient model exhibited a shorter APD90 (p < 0.001), a lower Smax (p < 0.001), mean DF (p = 0.012), PS number (p < 0.001), and a longer AF cycle length (AFCL, p = 0.011). Five V-AADs changed the electrophysiology in a dose dependent manner. AAD-induced AFCL lengthening (p < 0.001) and reductions in the CV (p = 0.033), peak DF (p < 0.001) and PS number (p < 0.001) were more significant in PITX2+/- deficient than wild-type AF. PITX2+/- deficient AF was easier to terminate with class IC AADs than the wild-type AF (p = 0.018). Conclusions The computational modeling-guided AAD test was feasible for evaluating the efficacy of multiple AADs in patients with AF. AF wave-dynamics and electrophysiological characteristics are different among the PITX2 deficient and the wild-type genotype models. BaselineChanges after AADClass ICClass IIIWild-typePITX2+/-p-valueWild-typePITX2+/-p-valueWild-typePITX2+/-p-valueWild-typePITX2+/-p-valueAPD90, (ms)243.7 ± 33.8184.4 ± 15.5<0.00138.2 ± 37.343.4 ± 56.20.223275.9 ± 43.5219.0 ± 39.2<0.001284.9 ± 32.8233.8 ± 71.4<0.001CV, (m/s)0.78 ± 0.320.70 ± 0.210.347-0.15 ± 0.18-0.20 ± 0.260.0330.63 ± 0.320.53 ± 0.300.0270.60 ± 0.360.43 ± 0.33<0.001Mean Smax0.787 ± 0.280.531 ± 0.18<0.0010.005 ± 0.260.115 ± 0.24<0.0010.828 ± 0.310.694 ± 0.320.0030.768 ± 0.320.608 ± 0.27<0.001Mean AFCL, (ms)146.96 ± 24.61164.78 ± 22.730.01122.62 ± 24.5537.92 ± 32.72<0.001165.44 ± 36.96190.85 ± 35.61<0.001169.05 ± 25.26203.35 ± 34.78<0.001Peak DF, (Hz)10.68 ± 2.9711.82 ± 3.340.211-2.98 ± 4.94-5.46 ± 4.66<0.00110.01 ± 4.397.23 ± 4.20<0.0016.30 ± 4.325.80 ± 4.070.301Mean DF, (Hz)6.80 ± 0.886.22 ± 0.710.012-1.95 ± 2.44-2.20 ± 1.990.2065.75 ± 1.784.53 ± 2.00<0.0014.14 ± 2.393.69 ± 2.000.077PS Number, (N)101086 ± 9608814150 ± 24778<0.001-59322 ± 99288-7409 ± 27856<0.00150579 ± 6523611568 ± 21868<0.00132951 ± 558643524 ± 8302<0.001PS Life Span, (ms)109.36 ± 113.90102.24 ± 226.640.889-24.87 ± 72.06-41.38 ± 126.350.073103.36 ± 180.6868.05 ± 162.790.14871.91 ± 141.8655.99 ± 217.970.454Table. Effects of AADs in the Wild-type and PITX2+/- Deficiency groupAbstract Figure. Wild-type vs. PITX2+/- baseline model

scholarly journals Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

2019 ◽  
Vol 85 (17) ◽  
Author(s):  
Zhu Jiang ◽  
Tengfei Niu ◽  
Xueqin Lv ◽  
Yanfeng Liu ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Efficiency ◽  
Molecular Engineering ◽  
Fine Tuning ◽  
Wild Type ◽  
Content Type ◽  
Chitosan Oligosaccharides ◽  
In The Wild ◽  
Secretory Production ◽  
Extracellular Activity

ABSTRACT Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5′ untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s−1 in the wild type to 7.56 μM s−1 in the mutant R157T. IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Mutations in the lrpE Gene of Ralstonia solanacearum Affects Hrp Pili Production and Virulence

2006 ◽  
Vol 19 (8) ◽  
pp. 884-895 ◽  
Author(s):  
Yukio Murata ◽  
Naoyuki Tamura ◽  
Kazuhiro Nakaho ◽  
Takafumi Mukaihara
Keyword(s):  
Cell Surface ◽  
Growth Phase ◽  
Ralstonia Solanacearum ◽  
Vascular System ◽  
Exponential Growth Phase ◽  
Bacterial Cells ◽  
Wild Type ◽  
Bacterial Surface ◽  
Bacterial Morphology ◽  
In The Wild

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the ΔlrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the ΔlrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the ΔlrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.

scholarly journals Iron-Dependent Cytochrome c1 Expression Is Mediated by the Status of Heme in Bradyrhizobium japonicum

2005 ◽  
Vol 187 (15) ◽  
pp. 5084-5089 ◽  
Author(s):  
Tao Gao ◽  
Mark R. O'Brian
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Aminolevulinic Acid ◽  
Iron Status ◽  
Covalent Binding ◽  
Dependent Manner ◽  
Wild Type ◽  
Prosthetic Group ◽  
Protein Levels ◽  
Genes Encoding ◽  
In The Wild

ABSTRACT The heme prosthetic group of heme proteins contains iron, which can be a limiting nutrient. Here, we show that cytochrome c 1 protein from Bradyrhizobium japonicum was strongly affected by the iron status, with low expression in cells grown under iron limitation. This control was not affected in mutants encoding the iron regulator Irr or Fur. Furthermore, cytochrome c 1 mRNA was not influenced by the iron status, suggesting control at a posttranscriptional step. Cytochrome c 1 protein levels were very low in mutants defective in the genes encoding δ-aminolevulinic acid (ALA) synthase and ferrochelatase, enzymes that catalyze the first and final steps of the heme biosynthetic pathway, respectively. Iron-dependent cytochrome c 1 expression was restored in the ALA synthase mutant by supplementation of the medium with the heme precursor ALA. Supplementation with heme resulted in high levels of cytochrome c 1 protein in the wild type and in both mutants, but expression was no longer iron dependent. Cytochrome c 1 is synthesized as a protein precursor fused with cytochrome b. A plasmid-borne construct encoding only cytochrome c 1 was expressed in an iron- and heme-dependent manner similar to that of the wild-type gene, indicating that control by those effectors is not linked to posttranslational processing of the fusion protein. Mutation of the cytochrome c 1 cysteines involved in covalent binding to heme nearly abolished immunodetectable protein. Thus, defects in heme synthesis or heme binding abrogate cytochrome c 1 accumulation, apparently due to protein degradation. We suggest that iron-dependent cytochrome c 1 expression is mediated by heme availability for heme protein formation

Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design

2020 ◽  
Vol 33 ◽  
Author(s):  
Subba Reddy Dodda ◽  
Nibedita Sarkar ◽  
Piyush Jain ◽  
Kaustav Aikat ◽  
Sudit S Mukhopadhyay
Keyword(s):  
Catalytic Activity ◽  
Protein Engineering ◽  
Aspergillus Fumigatus ◽  
Rational Design ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Substrate Affinity ◽  
Wild Type ◽  
Dynamics Simulations ◽  
Cellulose Binding

Abstract Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated from Aspergillus fumigatus (AfCel6A). Structure-based variants of AfCel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5–7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering.

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