Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis.
Role of the conserved Asn345 and Asn435 residues of Bacillus kaustophilus leucine aminopeptidase (BkLAP) was investigated by performing computer modeling and site-directed mutagenesis. Replacement of BkLAP Asn345 with Gln or Leu resulted in a dramatic reduction in enzymatic activity. A complete loss of the LAP activity was observed in Asn435 variants. Circular dichroism spectra were nearly identical for wild-type and all mutant enzymes, while measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in Asn345 and Asn435 replacements. Except for N435R and N435L, wild-type and other mutant enzymes showed a similar sensitivity towards temperature-induced denaturation. Computer modeling of the active-site structures of wild-type and mutant enzymes exhibits a partial or complete loss of the hydrogen bonding in the variants.
Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi 621, Taiwan.
Meng-Chun
MC
Chi
Ping-Lin
PL
Ong
Wen-Hwei
WH
Hsu
Yan-Hung
YH
Chen
Hsien-Bin
HB
Huang
Long-Liu
LL
Lin
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001216:Asparagine; D007931:Leucyl Aminopeptidase