Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design

Hamuro, Yuhi; Tajima, Katsuya; Matsumoto-Akanuma, Akiko; Sakamoto, Sayaka; Furukawa, Ryutaro; Yamagishi, Akihiko; Ohno, Naohito; Akanuma, Satoshi

doi:10.1016/j.jbiosc.2017.07.004

Cited by 10 publications

(7 citation statements)

References 47 publications

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“…Many groups succeeded in the design after the curation, for example, by removing sequences that are too short or long compared with STP 14 and by selecting sequences based on analysis of a phylogenetic tree. 52 In this study, we show that the utility of our proposed approach, which curates the library by reducing the E c value, is effective for achieving selection through the design and functional comparison of the artificial SDR-TDHs (FcTDH-N0, FcTDH-N1, and AncTDH). The curated library had the following features: Sequences sharing low-sequence identity (<about 50%) were omitted, and sequences belonging to five different classes were selected.…”

Section: ■ Discussionmentioning

confidence: 83%

“…A well-known approach is the curation of a sequence library. Many groups succeeded in the design after the curation, for example, by removing sequences that are too short or long compared with STP and by selecting sequences based on analysis of a phylogenetic tree . In this study, we show that the utility of our proposed approach, which curates the library by reducing the E c value, is effective for achieving selection through the design and functional comparison of the artificial SDR-TDHs (FcTDH-N0, FcTDH-N1, and AncTDH).…”

Section: Discussionmentioning

confidence: 83%

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Benchmark Analysis of Native and Artificial NAD⁺-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data

et al. 2018

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The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full-consensus design (FCD) and ancestral-sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial l-threonine 3-dehydrogenases (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1, and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH); the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5 °C higher than that of CnTDH, respectively, and the dissociation constants toward NAD of FcTDH-N1 and AncTDH were 2- and 7-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.

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Section: ■ Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 83%

Benchmark Analysis of Native and Artificial NAD⁺-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data

et al. 2018

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“…Proteins reconstructed in this way are often highly thermostable 29 – 32 . Moreover, by substituting putative ancestral amino acids into natural enzymes, the thermostability of a natural protein is highly likely to be increased without compromising the catalytic activity 33 – 37 .…”

Section: Introductionmentioning

confidence: 99%

Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

Furukawa

Toma

Yamazaki

et al. 2020

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Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

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“…Moreover, laccase has been produced by phylogenetic analysis of different Geobacillus strains using alignments with all Bacillus clusters. Phylogeny-based design makes up the construction of multiple amino acid sequence alignments (MSA) of homologous proteins, and phylogenetic tree creation was trailed by sequence inference at the deepest node (Hamuro et al 2017). Comparison of 16S rDNA sequence of strains with other associated B. amyloliquefaciens demonstrated that the 16S rDNA sequence of those strains had high similarity with Bacillus spp (99% individuality) (Lončar et al 2014).…”

Section: Discussionmentioning

confidence: 99%

Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia

et al. 2020

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Abstract. Mar WW, Rohman A, Muwafiqi NH, Laras GA, Agustina D, Asmarani O, Puspaningsih NNT. 2020. Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia. Biodiversitas 21: 2113-2118. Interpretation of phylogenetic trees is essential in understanding relationships between organisms, as well as their characteristics, bionetwork, and even their genomic and developmental ecology. The aim of this research is to analyze the phylogenetic bacteria producing laccase isolated from Gunung Pancar Bogor, Indonesia. Phylogenetic analysis of Geobacillus kaustophilus TP-02 producing laccase was performed using alignments with all Bacillus clusters. A phylogenetic tree was constructed by the neighbor-joining (NJ) method, using the maximum likelihood parameter. Laccase was purified using ammonium sulfate precipitation to 2.67-fold. The activity of crude laccase was determined to be 93.39 U/ml using 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Moreover, Geobacillus related to counting some Geobacillus genus such as Geobacillus thermocatenulatus, Geobacillus sp. MAS1, Geobacillus thermoleovorans, and Geobacillus zalihae, having similarity under 91% with comparison to the Geobacillus kaustophilius sequence in GenBank. Therefore, results show that G. kaustophilus TP-02 does not have any closeness with other Geobacillus genera, even though these species have the same ancestor.

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Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design

Cited by 10 publications

References 47 publications

Benchmark Analysis of Native and Artificial NAD⁺-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data

Benchmark Analysis of Native and Artificial NAD⁺-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data

Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia

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Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design

Cited by 10 publications

References 47 publications

Benchmark Analysis of Native and Artificial NAD+-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data

Benchmark Analysis of Native and Artificial NAD+-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data

Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

Short Communication: Preliminary phylogenetic analysis of bacteria producing laccase isolated from Gunung Pancar, Bogor, Indonesia

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Benchmark Analysis of Native and Artificial NAD⁺-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data

Benchmark Analysis of Native and Artificial NAD⁺-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data