Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.


Abstract

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.

Submission Details

ID: vFMPoNsk3

Submitter: Shu-Ching Ou

Submission Date: Feb. 1, 2019, 5:44 p.m.

Version: 1

Publication Details
Khodakarami A;Goodarzi N;Hoseinzadehdehkordi M;Amani F;Khodaverdian S;Khajeh K;Ghazi F;Ranjbar B;Amanlou M;Dabirmanesh B,Int J Biol Macromol (2018) Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity. PMID:29425870
Additional Information

Only values in Table 2 are included.

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2WSD 2010-09-29 1.6 Proximal mutations at the type 1 Cu site of CotA-laccase: I494A mutant
5ZLM 2018-05-16 1.7 Mutation in the trinuclear site of CotA-laccase: H491C mutant, PH 8.0
4AKO 2012-03-14 1.7 Mutations in the neighbourhood of CotA-laccase trinuclear site: E498L mutant
1GSK 2003-05-21 1.7 Crystal structure of CotA, an endospore coat protein from Bacillus subtilis
2X88 2010-09-22 1.8 Crystal Structure of HoloCotA
4Q8B 2015-04-29 1.91 The crystal structure of CotA laccase complexed with sinapic acid
4A66 2012-01-25 1.95 Mutations in the neighbourhood of CotA-laccase trinuclear site: D116A mutant
5ZLJ 2018-05-16 1.96 Crystal structure of CotA native enzyme, PH8.0
1W6L 2005-10-26 2.0 3D structure of CotA incubated with CuCl2
4A68 2012-01-25 2.0 Mutations in the neighbourhood of CotA-laccase trinuclear site: D116N mutant
4AKP 2012-03-14 2.0 Mutations in the neighbourhood of CotA-laccase trinuclear site: E498T mutant
2X87 2010-09-22 2.0 Crystal Structure of the reconstituted CotA
4AKQ 2012-03-14 2.1 Mutations in the neighbourhood of CotA-laccase trinuclear site: E498D mutant
4A67 2012-01-25 2.1 Mutations in the neighbourhood of CotA-laccase trinuclear site: D116E mutant
1W6W 2005-10-26 2.2 3D structure of CotA incubated with sodium azide
1W8E 2005-10-26 2.2 3D structure of CotA incubated with hydrogen peroxide
4YVU 2016-03-30 2.3 Crystal structure of CotA native enzyme in the acid condition, PH5.6
4YVN 2016-03-30 2.3 Crystal structure of CotA laccase complexed with ABTS at a novel binding site
4Q89 2015-04-29 2.31 Crystal Structure of the CotA native enzyme
3ZDW 2012-12-19 2.4 Substrate and dioxygen binding to the endospore coat laccase CotA from Bacillus subtilis
1OF0 2004-05-21 2.45 CRYSTAL STRUCTURE OF BACILLUS SUBTILIS COTA AFTER 1H SOAKING WITH ABTS
2BHF 2005-10-26 2.5 3D structure of the reduced form of CotA
5ZLL 2018-05-16 2.6 Mutation in the trinuclear site of CotA-laccase: H493C mutant, PH 8.0
5ZLK 2018-05-16 2.6 Mutation in the trinuclear site of CotA-laccase: H493A mutant, PH 8.0

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Spore coat protein A P07788 COTA_BACSU