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Applied Biochemistry and Biotechnology Jun 2016Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals....
Chiral α-hydroxy acids (AHAs) are rapidly becoming important synthetic building blocks, in particular for the production of pharmaceuticals and other fine chemicals. Chiral compounds of a variety of functionalities are now often derived using enzymes, and L-lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (bsLDH) has the potential to be employed for the industrial synthesis of chiral α-hydroxy acids. Despite the thorough characterization of this enzyme, generation of variants with high activity on non-natural substrates has remained difficult and therefore limits the use of bsLDH in industry. Here, we present the engineering of bsLDH using semi-rational design as a method of focusing screening in a small and smart library for novel biocatalysts. In this study, six mutant libraries were designed in an effort to expand the substrate range of bsLDH. The eight variants identified as having enhanced activity toward the selected α-keto acids belonged to the same library, which targeted two positions simultaneously. These new variants now may be useful biocatalysts for chiral synthesis of α-hydroxy acids.
Topics: Binding Sites; Escherichia coli; Geobacillus stearothermophilus; Hydroxy Acids; L-Lactate Dehydrogenase; Mutagenesis, Site-Directed; Mutation; Protein Engineering; Substrate Specificity
PubMed: 26852025
DOI: 10.1007/s12010-016-2007-x -
Biochemical Society Transactions Apr 1991
Topics: Chromatography, DEAE-Cellulose; Crystallization; Electrophoresis, Polyacrylamide Gel; Humans; Isoenzymes; L-Lactate Dehydrogenase; Myocardium
PubMed: 1889593
DOI: 10.1042/bst019219s -
Canadian Journal of Microbiology Mar 1995D-Lactate is readily used as a substrate for the growth of species of halophilic archaea belonging to the genera Haloferax and Haloarcula. L-Lactate was used by...
D-Lactate is readily used as a substrate for the growth of species of halophilic archaea belonging to the genera Haloferax and Haloarcula. L-Lactate was used by Haloferax species (Haloferax volcanii, Haloferax mediterranei) only when a substantial concentration of the D-isomer was also present in the medium. On the enzymatic level, considerable diversity was found in the lactate metabolism of the different representatives of the Halobacteriaceae. At least three types of lactate dehydrogenases were detected in halophilic archaea. A high level of activity of an NAD-linked enzyme was present constitutively in Haloarcula species, and a low level of activity was also detected in Haloferax mediterranei. NAD-independent lactate dehydrogenases, oxidizing L-lactate and D-lactate with 2,6-dichlorophenol-indophenol as electron acceptor, were detected in all nine species tested, but L-lactate dehydrogenase activity in Halobacterium species was very low, and Haloarcula species, which possess a high level of activity of NAD-linked lactate dehydrogenase, showed very low activities of both NAD-independent D- and L-lactate dehydrogenase. An inducible lactate racemase, displaying an unusually high pH optimum, was found in Haloferax volcanii. Lactate racemase activity was found constitutively in Haloarcula species, but no activity was detected in Halobacterium species and in Haloferax mediterranei.
Topics: Halobacteriaceae; L-Lactate Dehydrogenase; Lactates; Lactic Acid; NAD
PubMed: 7736359
DOI: 10.1139/m95-042 -
Clinical Biochemistry Mar 1999We report a case showing an atypical lactate dehydrogenase (LD) isoenzyme pattern involving deficiency only of LD-1 and LD-2 in serum and erythrocytes. LD activity in... (Review)
Review
OBJECTIVE
We report a case showing an atypical lactate dehydrogenase (LD) isoenzyme pattern involving deficiency only of LD-1 and LD-2 in serum and erythrocytes. LD activity in serum from this patient was extremely low, similar to complete LD-H deficiency, and also that in erythrocytes was low.
DESIGN
The DNA fragment containing exon 1 through 7 of the LD-H gene were amplified by PCR and directly sequenced. Total RNA was prepared from venous blood and the proportion of LD-H cDNA to total LD cDNA was semiquantified.
RESULTS
Genetic analysis by DNA sequencing detected a three base deletion (AAT) at codon 220 of exon 5, which caused a deletion of one asparagine. The present case did not show reduced LD-H expression at the mRNA level in whole blood. Residue 220 is involved in turning beta-J to alpha1-G and is not buried in the interior of the protein. The novel homozygous in-frame deletion mutation at codon 220 may cause a three-dimensional change of the subunit-binding domain.
Topics: Adult; Erythrocytes; Humans; Isoenzymes; L-Lactate Dehydrogenase; Male; Sequence Deletion
PubMed: 10211631
DOI: 10.1016/s0009-9120(98)00097-6 -
Annals of Clinical Biochemistry Mar 1999
Topics: Adolescent; Adult; Albuminuria; Amniotic Fluid; Ascitic Fluid; Chemistry, Clinical; Humans; Isoenzymes; L-Lactate Dehydrogenase; Male; Middle Aged; Pleural Effusion; Serum Albumin
PubMed: 10370750
DOI: 10.1177/000456329903600224 -
Research in Veterinary Science Sep 1987The total activity of lactate dehydrogenase (LDH) and the percentage distribution of its isoenzymes in the tissues and sera of clinically normal adult dogs are...
The total activity of lactate dehydrogenase (LDH) and the percentage distribution of its isoenzymes in the tissues and sera of clinically normal adult dogs are presented. Total LDH activity was greatest in skeletal muscle followed by heart muscle, kidney, small intestinal mucosa, liver, lung, pancreas and bone. Each tissue had a unique isoenzyme pattern and the proportions of the isoenzymes in serum suggested that liver is the source of normal serum LDH. The tissue isoenzyme patterns were similar to those obtained by other authors in human beings, horses, cattle, sheep and cats although in liver, differences between ruminants and monogastric animals including dogs were evident. The data presented provide a basis for the interpretation of serum LDH isoenzyme patterns in canine disease.
Topics: Animals; Dogs; Female; Isoenzymes; L-Lactate Dehydrogenase; Male; Tissue Distribution
PubMed: 3685636
DOI: No ID Found -
The Biochemical Journal Jun 19811. L-Lactate dehydrogenase from lettuce (Lactuca sativa) leaves was purified to electrophoretic homogeneity by affinity chromatography. 2. In addition to its...
1. L-Lactate dehydrogenase from lettuce (Lactuca sativa) leaves was purified to electrophoretic homogeneity by affinity chromatography. 2. In addition to its NAD(H)-dependent activity with L-lactate and pyruvate, the enzyme also catalyses the reduction of hydroxypyruvate and glyoxylate. The latter activities are not due to a contamination of the enzyme preparations with hydroxypyruvate reductase. 3. The enzyme shows allosteric properties that are markedly by the pH. 4. ATP is a potent inhibitor of the enzyme. The kinetic data suggest that the inhibition by ATP is competitive with respect to NADH at pH 7.0 and 6.2. The existence of regulatory binding sites for ATP and NADH is discussed. 5. Bivalent metal cations and fructose 6-phosphate relieve the ATP inhibition of the enzyme. 6. A function of leaf L-lactate dehydrogenase is proposed as a component of the systems regulating the cellular pH and/or controlling the concentration of reducing equivalents in the cytoplasm of leaf cells.
Topics: Adenosine Triphosphate; Cations, Divalent; Fructosephosphates; Kinetics; L-Lactate Dehydrogenase; NAD; Plants; Substrate Specificity
PubMed: 7316976
DOI: 10.1042/bj1950615 -
Bulletin of Experimental Biology and... Dec 2005The isoenzyme profile of lactate dehydrogenase in the cranial cervical sympathetic ganglion of rabbits was studied under normal conditions and during blockade of...
The isoenzyme profile of lactate dehydrogenase in the cranial cervical sympathetic ganglion of rabbits was studied under normal conditions and during blockade of nicotinic cholinergic synapses. Under normal conditions this profile was presented by 5 isoforms of the enzyme (lactate dehydrogenases 1, 2, 3, 4, and 5). Activity of H-isoforms prevailed. Blockade was accompanied by heterotropic allosteric inhibition of lactate dehydrogenase isoforms. H- and M-isoforms underwent simultaneous changes. Activity of H-isoforms sharply decreased. However, the ratio between lactate dehydrogenases 1 and 2 during complete or partial blockade did not differ from that observed in experiments with the intact ganglion. M-isoforms (lactate dehydrogenases 4 and 5) disappeared during partial blockade. Activity of hybrid lactate dehydrogenase 3 significantly decreased and was undetected during partial and complete blockade, respectively. Our results indicate that enzyme activity and isoenzyme profile of lactate dehydrogenase are determined by function of nicotinic synapses.
Topics: Animals; Isoenzymes; L-Lactate Dehydrogenase; Lactate Dehydrogenase 5; Nicotinic Agonists; Pipecolic Acids; Protein Isoforms; Rabbits; Superior Cervical Ganglion; Synapses; Synaptic Transmission
PubMed: 16848225
DOI: 10.1007/s10517-006-0055-x -
Biochemistry Nov 2018The malarial pathogen Plasmodium falciparum ( Pf) is a member of the Apicomplexa, which independently evolved a highly specific lactate dehydrogenase (LDH) from an...
The malarial pathogen Plasmodium falciparum ( Pf) is a member of the Apicomplexa, which independently evolved a highly specific lactate dehydrogenase (LDH) from an ancestral malate dehydrogenase (MDH) via a five-residue insertion in a key active site loop. PfLDH is widely considered an attractive drug target because of its unique active site. The conservation of the apicomplexan loop suggests that a precise insertion sequence was required for the evolution of LDH specificity. Aside from a single critical tryptophan, W107f, the functional and structural roles of residues in the loop are currently unknown. Here we show that the loop is remarkably robust to mutation, as activity is resilient to radical perturbations of both loop identity and length. Thus, alternative insertions could have evolved LDH specificity as long as they contained a tryptophan in the proper location. PfLDH likely has great potential to develop resistance to drugs designed to target its distinctive active site loop.
Topics: Amino Acid Sequence; Binding Sites; Catalytic Domain; Crystallography, X-Ray; L-Lactate Dehydrogenase; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Phylogeny; Plasmodium falciparum; Protein Conformation; Sequence Homology
PubMed: 30358994
DOI: 10.1021/acs.biochem.8b00913 -
Acta Crystallographica. Section F,... Aug 2014The thermostable D-lactate dehydrogenase from Lactobacillus jensenii (LjD-LDH) is a key enzyme for the production of the D-form of lactic acid from pyruvate concomitant...
The thermostable D-lactate dehydrogenase from Lactobacillus jensenii (LjD-LDH) is a key enzyme for the production of the D-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD(+). The polymers of lactic acid are used as biodegradable bioplastics. The LjD-LDH protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 28%(w/v) polyethylene glycol 400, 100 mM Tris-HCl pH 9, 200 mM magnesium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The crystal belonged to space group P3121, with unit-cell parameters a = b = 90.5, c = 157.8 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.58 Å(3) Da(-1), which corresponds to a solvent content of approximately 52.3%. The structure was solved by single-wavelength anomalous dispersion using a selenomethionine derivative.
Topics: Cloning, Molecular; Crystallization; Crystallography, X-Ray; Electrophoresis, Polyacrylamide Gel; L-Lactate Dehydrogenase; Lactobacillus
PubMed: 25084378
DOI: 10.1107/S2053230X14012606