An alternative allosteric regulation mechanism of an acidophilic l-lactate dehydrogenase from Enterococcus mundtii 15-1A.
A plant-derived Enterococcus mundtii 15-1A, that has been previously isolated from Brassica rapa L. subsp. nipposinica (L.H. Bailey) Hanelt var. linearifolia by our group, possesses two kinds of l-lactate dehydrogenase (l-LDH): LDH-1 and LDH-2. LDH-1 was activated under low concentration of fluctose-1,6-bisphosphate (FBP) at both pH 5.5 and 7.5. Although LDH-2 was also activated under the low concentration of FBP at pH 5.5, a high concentration of FBP is necessary to activate it at pH 7.5. The present study shows the crystal structures of the acidophilic LDH-2 in a complex with and without FBP and NADH. Although the tertiary structure of the ligands-bound LDH-2 is similar to that of the active form of other bacterial l-LDHs, the structure without the ligands is different from that of any other previously determined l-LDHs. Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit. At the N-terminal parts of the two regions, the ligands-bound form takes an α-helical structure, while the form without ligands displays more disordered and extended structures. A vacuum-ultraviolet circular dichroism analysis showed that the α-helix content of LDH-2 in solution is approximately 30% at pH 7.5, which is close to that in the crystal structure of the form without ligands. A D241N mutant of LDH-2, which was created by us to easily form an α-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Yasuyuki
Y
Matoba
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Masashi
M
Miyasako
Hiroshima Synchrotron Radiation Center, Hiroshima University, Kagamiyama 2-313, Higashi-Hiroshima 739-0046, Japan.
Koichi
K
Matsuo
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Kosuke
K
Oda
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Masafumi
M
Noda
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Fumiko
F
Higashikawa
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Takanori
T
Kumagai
Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Masanori
M
Sugiyama
Allosteric regulation; B., Bacillus; Bf., Bifidobacterium; CD, circular dichroism; Circular dichroism; Crystal structure; E., Escherichia; Ec., Enterococcus; FBP, fluctose-1,6-bisphosphate; LAB, lactic acid bacteria; LDH, lactate dehydrogenase; Lactic acid bacteria; Lb., Lactobacillus; Lc., Lactococcus; VUV, vacuum-ultraviolet; l-Lactate dehydrogenase; rms, root mean square