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Journal ArticleDOI

An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus casei L-lactate dehydrogenase activity induced by a single amino acid substitution.

01 Jan 2002-Protein Engineering (Oxford University Press)-Vol. 15, Iss: 1, pp 35-41
TL;DR: The role of His205 in the allosteric regulation of the enzyme is discussed on the basis of the known crystal structures of L-LDHs, indicating that His205 is also largely involved in the pH-dependent sensitivity of L.casei L- LDH to Fru(1,6)P(2).
Abstract: Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH) absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P 2 ] for its catalytic activity under neutral conditions, but exhibits marked catalytic activity in theabsence of Fru(1,6)P 2 under acidic conditions through the homotropic activation effect of substrate pyruvate. In this enzyme, a single amino acid replacement, i.e. that of His205 conserved in the Fru(1,6)P 2 -binding site of certain allosteric L-LDHs of lactic acid bacteria with Thr, did not induce a marked loss of the activation effect of Fru(1,6)P 2 or divalent metal ions, which are potent activators that improve the activation function of Fru(1,6)P 2 under neutral conditions. However, this replacement induced a great loss of the Fru(1,6)P 2 -independent activation effect of pyruvate or pyruvate analogs under acidic conditions, consequently indicating an absolute Fru(1,6)P 2 requirement for the enzyme activity. The replacement also induced a significant reduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P 2 , through a slight decrease and increase of the Fru(1,6)P 2 sensitivity under acidic and neutral conditions, respectively, indicating that His205 is also largely involved in the pH-dependent sensitivity of L.casei L-LDH to Fru(1,6)P 2 . The role of His205 in the allosteric regulation of the enzyme is discussed on the basis of the known crystal structures of L-LDHs.
Citations
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Journal ArticleDOI
TL;DR: The findings suggest that the heterologous expression of dnaKEco enhances the quality control of proteins and enzymes, resulting in improved growth and lactic acid fermentation at high temperature.
Abstract: The effects of nisin-induced dnaK expression in Lactococcus lactis were examined, and this expression was shown to improve stress tolerance and lactic acid fermentation efficiency. Using a nisin-inducible expression system, DnaK proteins from L. lactis (DnaKLla) and Escherichia coli (DnaKEco) were produced in L. lactis NZ9000. In comparison to a strain harboring the empty vector pNZ8048 (designated NZ-Vector) and one expressing dnaKLla (designated NZ-LDnaK), the dnaKEco-expressing strain, named NZ-EDnaK, exhibited more tolerance to heat stress at 40°C in GM17 liquid medium. The cell viability of NZ-Vector was reduced 4.6-fold after 6 h of heat treatment. However, NZ-EDnaK showed 13.5-fold increased viability under these conditions, with a very low concentration of DnaKEco production. Although the heterologous expression of dnaKEco did not effect DnaKLla production, heat treatment increased the DnaKLla level 3.5- and 3.6-fold in NZ-Vector and NZ-EDnaK, respectively. Moreover, NZ-EDnaK showed tolerance to multiple stresses, including 3% NaCl, 5% ethanol, and 0.5% lactic acid (pH 5.47). In CMG medium, the lactate yield and the maximum lactate productivity of NZ-EDnaK were higher than the corresponding values for NZ-Vector at 30°C. Interestingly, at 40°C, these values of NZ-EDnaK were not significantly different from the corresponding values for the control strain at 30°C. Lactate dehydrogenase (LDH) activity was also found to be stable at 40°C in the presence of DnaKEco. These findings suggest that the heterologous expression of dnaKEco enhances the quality control of proteins and enzymes, resulting in improved growth and lactic acid fermentation at high temperature.

84 citations

Journal ArticleDOI
20 Apr 2011-PLOS ONE
TL;DR: The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.
Abstract: Background Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal. Methodology/Principal Findings A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l−1) and high productivity (2.3 g l−1 h−1) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. Conclusions/Significance Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.

76 citations


Cites background from "An absolute requirement of fructose..."

  • ...Moreover, the amino acids involved in activation by fructose 1,6-bisphosphate (FDP) (Arg 173 and His 188) were also found in L-nLDH (Figure S1) [28]....

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Journal ArticleDOI
TL;DR: The role allostery plays in the functions of ATP-consuming molecular machines, bacterial chaperonin GroEL and molecular motors, is described and universal molecular principles governing allosteric signaling do not exist are drawn.
Abstract: Allosteric signaling in biological molecules, which may be viewed as specific action at a distance due to localized perturbation upon binding of ligands or changes in environmental cues, is pervasive in biology. Insightful phenomenological Monod, Wyman, and Changeux (MWC) and Koshland, Nemethy, and Filmer (KNF) models galvanized research in describing allosteric transitions for over five decades, and these models continue to be the basis for describing the mechanisms of allostery in a bewildering array of systems. However, understanding allosteric signaling and the associated dynamics between distinct allosteric states at the molecular level is challenging and requires novel experiments complemented by computational studies. In this review, we first describe symmetry and rigidity as essential requirements for allosteric proteins or multisubunit structures. The general features, with MWC and KNF as two extreme scenarios, emerge when allosteric signaling is viewed from an energy landscape perspective. To go beyond the general theories, we describe computational tools that are either based solely on multiple sequences or their structures to predict the allostery wiring diagram. These methods could be used to predict the network of residues that carry allosteric signals. Methods to obtain molecular insights into the dynamics of allosteric transitions are briefly mentioned. The utility of the methods is illustrated by applications to systems ranging from monomeric proteins in which there is little conformational change in the transition between two allosteric states to membrane bound G-protein coupled receptors and multisubunit proteins. Finally, the role allostery plays in the functions of ATP-consuming molecular machines, bacterial chaperonin GroEL and molecular motors, is described. Although universal molecular principles governing allosteric signaling do not exist, we can draw the following general conclusions from a survey of different systems. (1) Multiple pathways connecting allosteric states are highly heterogeneous. (2) Allosteric signaling is exquisitely sensitive to the specific architecture of the system, which implies that the capacity for allostery is encoded in the structure itself. (3) The mechanical modes that connect distinct allosteric states are robust to sequence variations. (4) Extensive investigations of allostery in Hemoglobin and, more recently GroEL, show that to a large extent a network of salt bridge rearrangements serves as allosteric switches. In both these examples the dynamical changes in the allosteric switches are related to function.

64 citations

Journal ArticleDOI
TL;DR: Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP).
Abstract: The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II phage library of C. thermocellu...

55 citations

Journal ArticleDOI
TL;DR: The results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate in Corynebacterium glutamicum at the industrial scale.
Abstract: We previously reported on the impacts of the overexpression of individual genes of the glycolytic pathway encoding glucokinase (GLK), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase (FBA) on D-lactate productivity in Corynebacterium glutamicum under oxygen-deprived conditions. Searching for synergies, in the current study, we simultaneously overexpressed the five glycolytic genes in a stepwise fashion to evaluate the effect of the cumulative overexpression of glycolytic genes on D-lactate production. Interestingly, the final D-lactate concentration markedly differed depending on whether or not the PFK encoding gene was overexpressed when combined with overexpressing other glycolytic genes. The simultaneous overexpression of the GLK, GAPDH, TPI, and FBA encoding genes led to the highest initial D-lactate concentration at 10 h. However, this particular recombinant strain dramatically slowed producing D-lactate when a concentration of 1300 mM was reached, typically after 32 h. In contrast, the strain overexpressing the PFK encoding gene together with the GLK, GAPDH, TPI, and FBA encoding genes showed 12.7 % lower initial D-lactate concentration at 10 h than that observed with the strain overexpressing the genes coding for GLK, GAPDH, TPI, and FBA. However, this recombinant strain continued to produce D-lactate after 32 h, reaching 2169 mM after a mineral salts medium bioprocess incubation period of 80 h. These results suggest that overexpression of the PFK encoding gene is essential for achieving high production of D-lactate. Our findings provide interesting options to explore for using C. glutamicum for cost-efficient production of D-lactate at the industrial scale.

46 citations


Cites background from "An absolute requirement of fructose..."

  • ...This observation can probably be ascribed to the upregulation of, on the one hand, the glycolytic enzymes, including glyceraldehyde phosphate dehydrogenase (GAPDH), triosephosphate isomerase (TPI), and phosphoglycerate kinase (PGK), and on the other hand enzymes of the anaplerotic pathway including phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) and LDH (Inui et al. 2007)....

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  • ...Indeed, the LDH activity in LPglc267/ pCRB215 (GLK, GAPDH, PFK, TPI, FBA) was higher than that in LPglc279/pCRB215 (GLK, GAPDH, TPI, FBA) both at 2 and 48 h (Fig....

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  • ...To test this possibility, we checked the enzyme activities of LDH and PFK, together with GLK, GAPDH, TPI, and FBA in LPglc267/pCRB215 (GLK, GAPDH, PFK, TPI, FBA) and LPglc279/pCRB215 (GLK, GAPDH, TPI, FBA) at 2 and 48 h in the bioprocess....

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  • ...Together with the LDH activity, the D-lactate yield in LPglc267/pCRB215 (GLK, GAPDH, PFK, TPI, FBA) was also higher than that in LPglc279/pCRB215 (GLK, GAPDH, TPI, FBA) with 90.0±1.4 and 83.8±1.9 %, respectively (Table 2)....

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  • ...In contrast, LPglc267/pCRB215 (GLK, GAPDH, PFK, TPI, FBA) should supply enough fructose-1,6-bisphohate for sufficient LDH activity even after 32 h in the bioprocess because of its increased PFK activity....

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References
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Journal ArticleDOI
15 Aug 1970-Nature
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.

232,912 citations


"An absolute requirement of fructose..." refers methods in this paper

  • ...The purity of the enzyme preparations was examined by SDS–PAGE according to Laemmli (Laemmli, 1970)....

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Journal Article
01 Jan 1970-Nature
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.

203,017 citations

Journal ArticleDOI
TL;DR: The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine, which is applied to mutations introduced via both oligonucleotides and error-prone polymerization.
Abstract: Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.

8,474 citations


"An absolute requirement of fructose..." refers methods in this paper

  • ...Preparation of the wild-type and mutant L.casei L-LDHs Oligodeoxynucleotide 5 -GAT GTT AGC AGT AGC TTG CCA TAC AGG-3 was purchased from Takara Syuzo and used to replace His205 with Thr. Site-directed mutagenesis was performed with a MUTA-GENE in vitro mutagenesis kit (Bio-RAD) according to Kunkel (Kunkel, 1985)....

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  • ...…the wild-type and mutant L.casei L-LDHs Oligodeoxynucleotide 5 -GAT GTT AGC AGT AGC TTG CCA TAC AGG-3 was purchased from Takara Syuzo and used to replace His205 with Thr. Site-directed mutagenesis was performed with a MUTA-GENE in vitro mutagenesis kit (Bio-RAD) according to Kunkel (Kunkel, 1985)....

    [...]

Journal ArticleDOI
TL;DR: "It is certain that all bodies whatsoever, though they have no sense, yet they have perception, and whether the body be alterant or alterec, evermore a perception precedeth operation; for else all bodies would be like one to another."

8,157 citations


"An absolute requirement of fructose..." refers background in this paper

  • ...In the case of the B.longum alosteric L-LDH, it has been reported that the allosteric transition can be assumed as two states, the inactive (T) and active (R) states, according to the concerted model demonstrated by Monod et al. (Monod et al., 1965), and the allosteric and catalytic sites communicate with each other through the quaternary structure change (Iwata et al., 1994; Fushinobu et al., 1996, 1998)....

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  • ...…transition can be assumed as two states, the inactive (T) and active (R) states, according to the concerted model demonstrated by Monod et al. (Monod et al., 1965), and the allosteric and catalytic sites communicate with each other through the quaternary structure change (Iwata et al., 1994;…...

    [...]

01 Jan 1979

766 citations