Recognition of phospholipids in Streptomyces phospholipase D.
To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly188 and Asp191 of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly188 and Asp191 are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.
Uesugi
Yoshiko
Y
Research Institute for Biological Sciences, Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Mori
Koichi
K
Arima
Jiro
J
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D015374:Biosensing Techniques; D020134:Catalytic Domain; D002855:Chromatography, Thin Layer; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D032621:DNA Shuffling; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D005998:Glycine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010739:Phospholipase D / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D010739:Phospholipase D; D005998:Glycine