Identification of Amino Acid Residues Essential for the Catalytic Reaction of<i>Bacillus kaustophilus</i>Leucine Aminopeptidase

Chi, Meng-Chun; Chou, Wei-Mou; Hsu, Wen-Hwei; Lin, Long-Liu

doi:10.1271/bbb.68.1794

Cited by 7 publications

(4 citation statements)

References 13 publications

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“…The proposed active-site residues are highly conserved in the M17 LAPs (Fig. 1A) and their importance has been evaluated using site-directed mutagenesis for EcPepA [15], Pseudomonas aeruginosa PhpA [18], tomato LAP-A [19], and Bacillus kaustophilus LAP (BkLAP) [20]. By sequence alignment, residues implicated for the zinc-coordination and catalytic reaction in BkLAP have been shown to be Lys265, Asp270, Lys277, Asp288, Asp347, Glu349, and Arg351 (Fig.…”

Section: Introductionmentioning

confidence: 99%

Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis

Chi

Ong

Hsu

et al. 2008

International Journal of Biological Macromolecules

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Section: Introductionmentioning

confidence: 99%

Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis

Chi

Ong

Hsu

et al. 2008

International Journal of Biological Macromolecules

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“…Our recent study showed that the amino acid sequence of Bacillus kaustophilus LAP (BkLAP) has a very high similarity to those of BlLAP and EcPep (Lin et al , 2004), and therefore it is expected that they might possess similar structural scaffolds. In fact, residues Lys‐250, Asp‐255, Lys‐262, Asp‐273, Asp‐332, Glu‐334 and Arg‐336 implicated in zinc coordination and catalytic reaction of BlLAP are conserved in BkLAP and the corresponding residues, Lys‐265, Asp‐270, Lys‐277, Asp‐288, Asp‐347, Glu‐349 and Arg‐351, have been shown to be important for the catalytic reaction of the enzyme (Chi et al , 2004). To understand further the structure–function relationship for BkLAP, a site‐directed mutagenesis strategy was pursued to characterize the role of Thr‐346 and Leu‐352 residues located in the vicinity of the enzyme active site.…”

Section: Introductionmentioning

confidence: 99%

Residues threonine 346 and leucine 352 are critical for the proper function of Bacillus kaustophilus leucine aminopeptidase

Chi¹,

Huang

Liu

et al. 2006

FEMS Microbiology Letters

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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.

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“…Based on X-ray structure and kinetic data of bovine lens enzyme, specific residues have been implicated in zinc-coordination and in catalysis [4][5][6]. The critical residues of bovine lens LAP are invariant in Escherichia coli PepA [7], tomato LAP-A [8], and Bacillus kaustophilus LAP (BkLAP) [9], suggesting that these enzymes might use a similar mechanism during the catalytic reaction.…”

Section: Introductionmentioning

confidence: 99%

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

Huang

Chi

Hsu

et al. 2005

Bioprocess Biosyst Eng

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The starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70 degrees C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K (m) value. Starch-binding assays showed that the K (d) and B (max) values for the fusion enzyme were 2.3 microM and 0.35 micromol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris-HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption-elution cycle with a purification of 11.4-fold.

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Identification of Amino Acid Residues Essential for the Catalytic Reaction ofBacillus kaustophilusLeucine Aminopeptidase

Cited by 7 publications

References 13 publications

Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis

Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis

Residues threonine 346 and leucine 352 are critical for the proper function of Bacillus kaustophilus leucine aminopeptidase

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

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