Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling.
To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.
Negishi
Tomofumi
T
Research Institute for Biological Sciences, Okayama (RIBS), 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Mukaihara
Takafumi
T
Mori
Koichi
K
Nishikido
Hiroko
H
Kawasaki
Yuko
Y
Aoki
Hiroyuki
H
Kodama
Michiko
M
Uedaira
Hatsuho
H
Uesugi
Yoshiko
Y
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*
D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D010739:Phospholipase D