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An alternative allosteric regulation mechanism of an acidophilic l-lactate dehydrogenase from Enterococcus mundtii 15-1A.

Matoba Y, Miyasako M, Matsuo K, Oda K, Noda M, Higashikawa F, Kumagai T, Sugiyama M - FEBS Open Bio (2014)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Sequence alignment of the Ec. mundtii LDH-2 with other l-LDHs from various LAB species. Sequence alignment was done using the ClustalX program [44]. Em-LDH-2, Ec. mundtii LDH-2; Ef-LDH-2, Ec. faecalis LDH-2; Ll-LDHB, Lc. lactis LDHB; Lp-LDH, Lb. pentosusl-LDH; Bl-LDH, Bf. longuml-LDH; Lc-LDH, Lb. caseil-LDH; Em-LDH-1, Ec. mundtii LDH-1; Ef-LDH-1, Ec. faecalis LDH-1; Ll-LDH, Lc. lactis LDH. The fully conserved residues in all sequences are marked with black shading. The residues conserved among alternative l-LDHs (Em-LDH-2, Ef-LDH-2, and Ll-LDHB) are marked with gray shading. In addition, the residues conserved among general allosteric l-LDHs (Bl-LDH, Lc-LDH, Em-LDH-1, Ef-LDH-1, and Ll-LDH) are also marked with gray shading. The residues of l-LDHs are numbered according to the N-system for vertebrate l-LDHs proposed by Eventoff et al. [45].
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f0005: Sequence alignment of the Ec. mundtii LDH-2 with other l-LDHs from various LAB species. Sequence alignment was done using the ClustalX program [44]. Em-LDH-2, Ec. mundtii LDH-2; Ef-LDH-2, Ec. faecalis LDH-2; Ll-LDHB, Lc. lactis LDHB; Lp-LDH, Lb. pentosusl-LDH; Bl-LDH, Bf. longuml-LDH; Lc-LDH, Lb. caseil-LDH; Em-LDH-1, Ec. mundtii LDH-1; Ef-LDH-1, Ec. faecalis LDH-1; Ll-LDH, Lc. lactis LDH. The fully conserved residues in all sequences are marked with black shading. The residues conserved among alternative l-LDHs (Em-LDH-2, Ef-LDH-2, and Ll-LDHB) are marked with gray shading. In addition, the residues conserved among general allosteric l-LDHs (Bl-LDH, Lc-LDH, Em-LDH-1, Ef-LDH-1, and Ll-LDH) are also marked with gray shading. The residues of l-LDHs are numbered according to the N-system for vertebrate l-LDHs proposed by Eventoff et al. [45].

Mentions: The sequence identity between the Ec. mundtii LDH-1 and LDH-2 is 44.9% (Fig. 1). However, the Ec. mundtii LDH-1 shows a high sequence identity with the Ec. faecalis LDH-1 (86.2%) and the Lc. lactis LDH (68.7%), whereas the Ec. mundtii LDH-2 shows a high sequence identity with the Ec. faecalis LDH-2 (65.0%) and the Lc. lactis LDHB (71.0%). These results indicate that LDH-1 mainly plays a role in l-lactate production in Ec. mundtii, while LDH-2 plays an additional role.


An alternative allosteric regulation mechanism of an acidophilic l-lactate dehydrogenase from Enterococcus mundtii 15-1A.

Matoba Y, Miyasako M, Matsuo K, Oda K, Noda M, Higashikawa F, Kumagai T, Sugiyama M - FEBS Open Bio (2014)

Sequence alignment of the Ec. mundtii LDH-2 with other l-LDHs from various LAB species. Sequence alignment was done using the ClustalX program [44]. Em-LDH-2, Ec. mundtii LDH-2; Ef-LDH-2, Ec. faecalis LDH-2; Ll-LDHB, Lc. lactis LDHB; Lp-LDH, Lb. pentosusl-LDH; Bl-LDH, Bf. longuml-LDH; Lc-LDH, Lb. caseil-LDH; Em-LDH-1, Ec. mundtii LDH-1; Ef-LDH-1, Ec. faecalis LDH-1; Ll-LDH, Lc. lactis LDH. The fully conserved residues in all sequences are marked with black shading. The residues conserved among alternative l-LDHs (Em-LDH-2, Ef-LDH-2, and Ll-LDHB) are marked with gray shading. In addition, the residues conserved among general allosteric l-LDHs (Bl-LDH, Lc-LDH, Em-LDH-1, Ef-LDH-1, and Ll-LDH) are also marked with gray shading. The residues of l-LDHs are numbered according to the N-system for vertebrate l-LDHs proposed by Eventoff et al. [45].
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219987&req=5

f0005: Sequence alignment of the Ec. mundtii LDH-2 with other l-LDHs from various LAB species. Sequence alignment was done using the ClustalX program [44]. Em-LDH-2, Ec. mundtii LDH-2; Ef-LDH-2, Ec. faecalis LDH-2; Ll-LDHB, Lc. lactis LDHB; Lp-LDH, Lb. pentosusl-LDH; Bl-LDH, Bf. longuml-LDH; Lc-LDH, Lb. caseil-LDH; Em-LDH-1, Ec. mundtii LDH-1; Ef-LDH-1, Ec. faecalis LDH-1; Ll-LDH, Lc. lactis LDH. The fully conserved residues in all sequences are marked with black shading. The residues conserved among alternative l-LDHs (Em-LDH-2, Ef-LDH-2, and Ll-LDHB) are marked with gray shading. In addition, the residues conserved among general allosteric l-LDHs (Bl-LDH, Lc-LDH, Em-LDH-1, Ef-LDH-1, and Ll-LDH) are also marked with gray shading. The residues of l-LDHs are numbered according to the N-system for vertebrate l-LDHs proposed by Eventoff et al. [45].
Mentions: The sequence identity between the Ec. mundtii LDH-1 and LDH-2 is 44.9% (Fig. 1). However, the Ec. mundtii LDH-1 shows a high sequence identity with the Ec. faecalis LDH-1 (86.2%) and the Lc. lactis LDH (68.7%), whereas the Ec. mundtii LDH-2 shows a high sequence identity with the Ec. faecalis LDH-2 (65.0%) and the Lc. lactis LDHB (71.0%). These results indicate that LDH-1 mainly plays a role in l-lactate production in Ec. mundtii, while LDH-2 plays an additional role.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus