Remarkable enhancement in PLD activity from Streptoverticillium cinnamoneum by substituting serine residue into the GG/GS motif.
The gene that encodes phospholipase D (PLD) from Streptoverticillium cinnamoneum contains three consensus regions (Region I, II and IV as shown in Fig. 1A) that are conserved among the PLD superfamily. The glycine-glycine (GG) motif in Region I and the glycine-serine (GS) motif in Region IV are also conserved in the PLD superfamily. These (GG and GS) motifs are located 7 residues downstream from each HKD motif. In an investigation of fifteen GG/GS motif mutants, generated as fusion proteins with maltose-binding protein (MBP-PLDs), three highly active mutants were identified. Three of the mutants (G215S, G216S, and G216S-S489G) contained a serine residue in the GG motif, and exhibited approximately a 9-27-fold increased transphosphatidylation activity to DPPC compared with recombinant wild type MBP-PLD. When heat stability was compared between three mutants and the recombinant wild type, only G216S-S489G showed heat labile properties. It appears that the 489th serine residue in the GS motif also contributes to the thermal stability of the enzyme. In addition, the GG/GS motif was very close to the active center residue, including two HKD motifs, as shown by computer modeling. The findings suggest that the GG/GS motif of PLD is a key motif that affects catalytic function and enzymatic stability.
Ogino
Chiaki
C
The Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan. ochiaki@t.kanazawa-u.ac.jp
Daido
Hidenori
H
Ohmura
Yuka
Y
Takada
Namiko
N
Itou
Yoshiki
Y
Kondo
Akihiko
A
Fukuda
Hideki
H
Shimizu
Nobuaki
N
D020816:Amino Acid Motifs; D002942:Circular Dichroism; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics* / Q000378:metabolism*; D013306:Streptomycetaceae / Q000201:enzymology* / Q000235:genetics
D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D012694:Serine; D010739:Phospholipase D; D005998:Glycine