Residues threonine 346 and leucine 352 are critical for the proper function of Bacillus kaustophilus leucine aminopeptidase.
The importance of Thr-346 and Leu-352 residues in Bacillus kaustophilus leucine aminopeptidase (BkLAP) was explored by site-directed mutagenesis. The impact of substitutions at these positions was evaluated with His6-BkLAP fusion proteins expressed in Escherichia coli. Substitution of Thr-346 with Tyr, Arg, and Leu, respectively, resulted in a dramatic reduction in LAP activity. A complete loss of activity was observed in L352E and L352R variants with the exception of L352 V, which retained approximately 60% of the wild-type activity. Zinc content analysis and protein modeling suggested that Thr-346 and Leu-352 of BkLAP play a role in maintaining the coordination environment for the zinc-binding residues.
Chi
Meng-Chun
MC
Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi, Taiwan.
Huang
Hsien-Ben
HB
Liu
Jai-Shin
JS
Wang
Wen-Ching
WC
Liang
Wan-Chi
WC
Lin
Long-Liu
LL
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000737:chemistry*; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry*; D015032:Zinc / Q000378:metabolism
D013912:Threonine; D007931:Leucyl Aminopeptidase; D007930:Leucine; D015032:Zinc