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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

Molecular surfaces of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. The surfaces of the Ec. mundtii LDH-2 in the active and inactive states are shown in a and b, respectively. The surfaces of the Bf. longuml-LDH in the active and inactive states [9] are shown in c and d, respectively. Each subunit in the tetramer is colored the same as in Fig. 3. In a, c and d, four NADH and two FBP molecules bound to the enzyme are shown in the stick model colored orange. In c, four oxamate (a pyruvate analog) molecules bound to the active-state Bf. longuml-LDH are shown in the stick model colored blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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f0035: Molecular surfaces of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. The surfaces of the Ec. mundtii LDH-2 in the active and inactive states are shown in a and b, respectively. The surfaces of the Bf. longuml-LDH in the active and inactive states [9] are shown in c and d, respectively. Each subunit in the tetramer is colored the same as in Fig. 3. In a, c and d, four NADH and two FBP molecules bound to the enzyme are shown in the stick model colored orange. In c, four oxamate (a pyruvate analog) molecules bound to the active-state Bf. longuml-LDH are shown in the stick model colored blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: It has been suggested that the side-chain orientation of the Arg171 residue is important for discriminating between inactive and active states. The side chain of the Arg171 residue of the Ec. mundtii LDH-2, which seems to be important for the binding of pyruvate, points toward the pyruvate-binding site (Fig. 6a), as seen in the active state of other bacterial l-LDHs. Furthermore, the quaternary structure of the ligands-bound LDH-2 is similar to that of the other bacterial l-LDHs in the active state, in which a compact tetramer is formed by many inter-subunit interactions (Fig. 7a and c). Therefore, we defined the quaternary structure of the ligands-bound LDH-2 as an active state.


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)

Molecular surfaces of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. The surfaces of the Ec. mundtii LDH-2 in the active and inactive states are shown in a and b, respectively. The surfaces of the Bf. longuml-LDH in the active and inactive states [9] are shown in c and d, respectively. Each subunit in the tetramer is colored the same as in Fig. 3. In a, c and d, four NADH and two FBP molecules bound to the enzyme are shown in the stick model colored orange. In c, four oxamate (a pyruvate analog) molecules bound to the active-state Bf. longuml-LDH are shown in the stick model colored blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0035: Molecular surfaces of the Ec. mundtii LDH-2 and the Bf. longuml-LDH. The surfaces of the Ec. mundtii LDH-2 in the active and inactive states are shown in a and b, respectively. The surfaces of the Bf. longuml-LDH in the active and inactive states [9] are shown in c and d, respectively. Each subunit in the tetramer is colored the same as in Fig. 3. In a, c and d, four NADH and two FBP molecules bound to the enzyme are shown in the stick model colored orange. In c, four oxamate (a pyruvate analog) molecules bound to the active-state Bf. longuml-LDH are shown in the stick model colored blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: It has been suggested that the side-chain orientation of the Arg171 residue is important for discriminating between inactive and active states. The side chain of the Arg171 residue of the Ec. mundtii LDH-2, which seems to be important for the binding of pyruvate, points toward the pyruvate-binding site (Fig. 6a), as seen in the active state of other bacterial l-LDHs. Furthermore, the quaternary structure of the ligands-bound LDH-2 is similar to that of the other bacterial l-LDHs in the active state, in which a compact tetramer is formed by many inter-subunit interactions (Fig. 7a and c). Therefore, we defined the quaternary structure of the ligands-bound LDH-2 as an active state.

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