l‐Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium

Abstract

Heat-stable and fructose-I ,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1 .I .27) has been purified from an extremely thermophilic bacterium, Thermus caldophilus GK24 [Taguchi, H., Yamashita, M., Matsuzawa, H. and Ohta, T. (1982) J. Biochem. (Tokyo) 91,. N-terminal sequence analysis of the first 34 amino acids of the enzyme indicates that the N-terminal arm region (first 1-20 residues) known for the vertebrate L-lactate dehydrogenases is completely missing in the T. caldophilus enzyme, while there is a high homology of sequence between the regions which are considered to be part of the NAD-binding domain. The C-terminal amino acid of the enzyme was phenylalanine. Analysis of the amino acid composition showed that T. caldophilus enzyme contained much more arginine and fewer lysine than other bacterial and vertebrate L-lactate dehydrogenases.On modification reaction with 2,3-butanedione in the presence of NADH and oxamate, an enhanced activity of the T. caldophilus L-lactate dehydrogenase was obtained independently of fructose 1,6-bisphosphate, and the modified enzyme was desensitized to fructose 1,6-bisphosphate. Amino acid analysis indicated that such a desensitization in the active state was caused by the modification of only one arginine residue per the enzyme subunit. Desensitization of the enzyme was inhibited in the presence of fructose 1,6-bisphosphate. A similar desensitization was observed using 1,2-~yclohexanedione instead of 2,3-butanedione. The enzyme was irreversibly modified with 2,3-butanedione and characterized. The irreversibly modified enzyme also showed an enhanced activity independently of fructose 1,6-bisphosphate, and its pyruvate saturation curve was similar to that of the native enzyme measured in the presence of fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate, which increases the thermostability of the native enzyme, did not affect that of the modified enzyme, while thermostability of the modified enzyme slightly decreased. Amino acid analysis indicated that only the arginine content was decreased by the modification. These results show that arginine residue(s) exist in the binding site for fructose 1,6-bisphosphate on the enzyme, and that the arginine residue(s) play some important role in the allosteric regulation of the enzyme activity.The protein structure and function of L-lactate dehydrogenase (EC 1 .I .1.27) have been studied in great detail for the enzyme of vertebrates [I] We previously reported on L-lactate dehydrogenase from an extremely thermophilic bacterium, Thermus caldophilus GK24, which has not only extreme thermostability but also an allosteric nature dependent on fructose 1,6-bisphosphate [19]. Because of the extremely high stability, T . caldophilus enzyme seems to have an advantage for studying the regulatory mechanism of the enzyme using a chemical modification method and for analyzing the thermostabilizing mechanism of the enzyme through comparison of structures between this enzyme and others. In this paper, we describe the activation and desensiti...

Identification of an allosteric site residue of a fructose 1,6‐bisphosphate‐dependent L‐lactate dehydrogenase of Thermus caldophilus GK24: production of a non‐allosteric form by protein engineering

“…Isolation of a peptide containing a modified Arg residue The T. caldophilus LDH was purified as described previously [14]. 5 mg of the enzyme was dissolved in 5 ml of 50 mM sodium borate buffer (pH 7.5) containing 10 mM NADH and 50 mM oxamate in the absence and presence of 10 mM Fru-1,6-Pz, and then 2,3-butanedione was added at 2.5 mM, as described previously [17]. The modification reaction was car-out at 30°C 30 min.…”

“…We studied the mechanism underlying allosteric regulation of the LDH by means of 'H NMR [16] and chemical modification [17]. The LDH can be desensitized to Fru-1,6-P2 in the activated state through Arg-specific chemical modification in the presence of NADH and oxamate, and the absence of Fru-1,6-P2 [ 171.…”

Amino Acid Residues in the Allosteric Site ofl-Lactate Dehydrogenase fromBifidobacterium longum

“…A crude extract was obtained from 600 g (wet weight) of cells by the method described by Taguchi et al 3 ) Tris-HCI buffer (20 mM, pH 7.0) was used· throughout the operations described below. From the supernatant of 40% saturation of ammonium sulfate, an active fraction was separated by a column of Butyl-Toyopearl 650 M (Tosoh Corp.) with decreasing linear gradient of 40 to 0% saturation of ammonium sulfate.…”

“…But, the concentration of FBP required for half-maximum activation for the enzyme was quite low compared to the values obtained in the case of other bacterial LDHs (0.6,,-,4.6 pM). 3,12) Peptides obtained by proteolysis were separated into about 16 peaks by reverse phase chromatography. The Nterminal sequence of peak-16 was GATNYAIGM-SGVDIIEAVLHDTNRILPVSSM.…”

Role of Histidine 188 in Fructose 1,6-Bisphosphate- and Divalent Cation-RegulatedL-Lactate Dehydrogenase ofLactobacillus casei

“…7) Allosteric L-LDHs, which are usually activated by fructose 1,6-bisphosphate [Fru(l ,6)P 2J, have been found mostly in bacterial cells and studied in detail, together with vertebrate non-allosteric L-LDHs. 8 • 9 ) Mechanisms underlying the regulation have been extensively studied in the cases of Lactobacillus casei, [10][11][12][13] Thermus caldophilus, [14][15][16][17][18][19] Bacillus stearothermophilus, [20][21][22][23] and Bifidobacterium longum 24 -27) allosteric L-LDHs. Among the above allosteric L-LDHs, the Lactobacillus enzyme uniquely requires certain divalent metal ions besides Fru( 1 ,6)P 2 at neutral pH, constituting a subclass of allosteric L-LDH that requires metal ions,28) but the machinery underlying such a unique regulation has not been clearly understood mostly because of a lack of analysis based on protein engineering, which has been very effective in the cases of the other * Corresponding author.…”