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

Arai, Kazuhito; Hishida, Atsushi; Ishiyama, Mariko; Kamata, Takeo; Uchikoba, Hiroyuki; Fushinobu, Shinya; Matsuzawa, Hiroshi; Taguchi, Hayao

doi:10.1093/protein/15.1.35

Cited by 25 publications

(27 citation statements)

References 34 publications

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“…Multiple alignments with other reported l -nLDHs showed that all catalytically important residues (Arg 109, Asp 168, Arg 171, and His 195) were conserved in the B. coagulans SDM l -nLDH (Figure S1) [27]. 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]. The essential residues of d -nLDH (Arg 235, Glu 264, and His 296) were also conserved in the B. coagulans SDM d -nLDH (Figure S2) [29], [30].…”

Section: Resultsmentioning

confidence: 88%

Efficient Conversion of Phenylpyruvic Acid to Phenyllactic Acid by Using Whole Cells of Bacillus coagulans SDM

Zheng

Gao

et al. 2011

PLoS ONE

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BackgroundPhenyllactic 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 FindingsA 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/SignificanceTaking 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.

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Section: Resultsmentioning

confidence: 88%

Efficient Conversion of Phenylpyruvic Acid to Phenyllactic Acid by Using Whole Cells of Bacillus coagulans SDM

Zheng

Gao

et al. 2011

PLoS ONE

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“…Recombinant LCLDH was expressed in Escherichia coli MV1184, and purified according to previous reports 23, 31. Type I crystals were prepared by the hanging drop vapor diffusion method at 25°C in 100 m M sodium citrate buffer (pH 4.4), 200 m M potassium sodium tartrate, and 1.6 M ammonium sulfate, and then transferred to a solution containing 1.8 M ammonium sulfate and 30% glycerol before flash‐cooling.…”

Section: Methodsmentioning

confidence: 99%

“…Oligodeoxynucleotide 5′‐GGT TAA CGT TGA TGC ACA ATC AGT CCA CGC TTA CAT CAT GGG‐3′ was used to replace Arg185 with Gln. Site‐directed mutagenesis was performed using the Gene Editor System from Promega, and expression and purification of the mutant LCLDH were performed by the same procedures as those used for the wild‐type enzyme 23…”

Section: Methodsmentioning

confidence: 99%

“…At pH 7.0, on the other hand, the enzyme absolutely depends on FBP, and requires more than 10 4 ‐ to 10 5 ‐fold higher concentration of FBP than at pH 5.0 for the catalytic reaction, though the activation function of FBP is markedly improved by certain divalent cations such as Mn 2+ . Site‐specific replacement of His205 with Thr markedly reduces the pH‐dependence of the allosteric activation through reductions of both the homotropic and heterotropic activation effects under acidic conditions, although the crucial function of His205 in the allosteric regulation is unknown 23…”

Section: Introductionmentioning

confidence: 99%

“…1), indicating that the two Lactobacillus enzymes diverged recently and acquired their distinct properties through minor structural changes during evolution. We are comparatively studying LCLDH and LPLDH to understand their structure‐function relationships,23, 24, 31–33 and have already determined the crystal structure of LPLDH at 2.3 Å resolution 33. On the other hand, however, the crystal structure of LCLDH has only been determined at poor resolution (3.0 Å) and with a poor crystallographic R‐factor (37.4%) 34.…”

Section: Introductionmentioning

confidence: 99%

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Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

et al. 2009

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Lactobacillus casei L-lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6-bisphosphate (FBP), respectively, and exhibits unusually high pH-dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 A resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q-axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra-helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L-lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P-axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q-axis related dimer through the rigid NAD-binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH-dependent allosteric equilibrium.

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Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation

Tsuge

Yamamoto

Kato

et al. 2015

Appl Microbiol Biotechnol

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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.

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

Cited by 25 publications

References 34 publications

Efficient Conversion of Phenylpyruvic Acid to Phenyllactic Acid by Using Whole Cells of Bacillus coagulans SDM

Efficient Conversion of Phenylpyruvic Acid to Phenyllactic Acid by Using Whole Cells of Bacillus coagulans SDM

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation

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