Limits...
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

Inter-subunit interactions around the Arg171 residue found in the Ec. mundtii LDH-2. The structures around the Arg171 residue of subunit A in the active state and of subunits A and B in the inactive state are shown in a, c, and d, respectively. Ribbon models of subunits A and B are shown in magenta and cyan, respectively. The residues Lys58, Glu60, Glu62, Asp65, Asp68, Trp72, Gly73, and Asn76 in one subunit and Thr166, Thr170, Arg171, Lys174, Glu175, Lys243, Thr246, Tyr248, and Gly249 in the other subunit are shown in the stick model. Hydrogen bonds are shown by broken lines. View directions are opposite in a and b.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4219987&req=5

f0040: Inter-subunit interactions around the Arg171 residue found in the Ec. mundtii LDH-2. The structures around the Arg171 residue of subunit A in the active state and of subunits A and B in the inactive state are shown in a, c, and d, respectively. Ribbon models of subunits A and B are shown in magenta and cyan, respectively. The residues Lys58, Glu60, Glu62, Asp65, Asp68, Trp72, Gly73, and Asn76 in one subunit and Thr166, Thr170, Arg171, Lys174, Glu175, Lys243, Thr246, Tyr248, and Gly249 in the other subunit are shown in the stick model. Hydrogen bonds are shown by broken lines. View directions are opposite in a and b.

Mentions: In summary, the N-terminal parts of the two regions (Val54–Lys75 and Lys233–Gly249) form the α-helices in the active state LDH-2, while the parts take more disordered and extended structures in the compact inactive state LDH-2. It should be noted that the residues Lys233–Gly249 of one subunit are positioned near the residues Val54–Lys75 of the X-axis-related subunit (Fig. 8a and b), indicating that the binding of NADH to one subunit facilitates the formation of the pyruvate-binding pocket in the X-axis-related subunit.


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)

Inter-subunit interactions around the Arg171 residue found in the Ec. mundtii LDH-2. The structures around the Arg171 residue of subunit A in the active state and of subunits A and B in the inactive state are shown in a, c, and d, respectively. Ribbon models of subunits A and B are shown in magenta and cyan, respectively. The residues Lys58, Glu60, Glu62, Asp65, Asp68, Trp72, Gly73, and Asn76 in one subunit and Thr166, Thr170, Arg171, Lys174, Glu175, Lys243, Thr246, Tyr248, and Gly249 in the other subunit are shown in the stick model. Hydrogen bonds are shown by broken lines. View directions are opposite in a and b.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0040: Inter-subunit interactions around the Arg171 residue found in the Ec. mundtii LDH-2. The structures around the Arg171 residue of subunit A in the active state and of subunits A and B in the inactive state are shown in a, c, and d, respectively. Ribbon models of subunits A and B are shown in magenta and cyan, respectively. The residues Lys58, Glu60, Glu62, Asp65, Asp68, Trp72, Gly73, and Asn76 in one subunit and Thr166, Thr170, Arg171, Lys174, Glu175, Lys243, Thr246, Tyr248, and Gly249 in the other subunit are shown in the stick model. Hydrogen bonds are shown by broken lines. View directions are opposite in a and b.
Mentions: In summary, the N-terminal parts of the two regions (Val54–Lys75 and Lys233–Gly249) form the α-helices in the active state LDH-2, while the parts take more disordered and extended structures in the compact inactive state LDH-2. It should be noted that the residues Lys233–Gly249 of one subunit are positioned near the residues Val54–Lys75 of the X-axis-related subunit (Fig. 8a and b), indicating that the binding of NADH to one subunit facilitates the formation of the pyruvate-binding pocket in the X-axis-related subunit.

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