Crystal structure of β‐galactosidase from <i>Bacillus circulans </i><scp>ATCC</scp> 31382 (BgaD) and the construction of the thermophilic mutants

Ishikawa, Kazuhiko; Kataoka, Mikio; Yanamoto, Toshiaki; Nakabayashi, Makoto; Watanabe, Masahiro; Ishihara, Satoru; Yamaguchi, Shotaro

doi:10.1111/febs.13298

Cited by 46 publications

(36 citation statements)

References 39 publications

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“…It is a large protein, and its shortest active fragment used in this work was ~100 kDa (termed BgaD-D; see Online Methods) 10 . Its three-dimensional structure in the apo form has very recently been reported 11 after the completion of the experiments described here. It has low homology, even at positions surrounding the active site, to well-characterized members—such as E. coli LacZ (25% sequence identity)—of the GH2 glycosidase family to which BgaD belongs 7 .…”

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confidence: 60%

Monobody-mediated alteration of enzyme specificity

et al. 2015

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Current methods for engineering enzymes modify enzymes themselves and require a detailed mechanistic understanding or a high-throughput assay. Here, we describe a new approach where catalytic properties are modulated with synthetic binding proteins, termed monobodies, directed to an unmodified enzyme. Using the example of a β-galactosidase from Bacillus circulans, we efficiently identified monobodies that restricted its substrates for its transgalactosylation reaction and selectively enhanced the production of small oligosaccharide prebiotics.

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confidence: 60%

Monobody-mediated alteration of enzyme specificity

et al. 2015

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“…The other transcription factors resulting from the DALI search showing high degree of structural homology to MyRF DBD include members of STAT 17 , Runx 18 . Some other Ig-fold proteins, such as the Cholesterol-binding Protein 19, 20 , ATP binding part of ABC Transporter 21 and sugar-binding domain of β-galactosidase 22 , have even higher score in the DALI search than p53, STAT and Runx, but they do not belong to the transcription factor family.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the DNA-binding domain of Myelin-gene Regulatory Factor

Zhen

et al. 2017

Sci Rep

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Myelin-gene Regulatory Factor (MyRF) is one of the master transcription factors controlling myelin formation and development in oligodendrocytes which is crucial for the powerful brain functions. The N-terminal of MyRF, which contains a proline-rich region and a DNA binding domain (DBD), is auto-cleaved from the ER membrane, and then enters the nucleus to participate in transcription regulation of the myelin genes. Here we report the crystal structure of MyRF DBD. It shows an Ig-fold like architecture which consists of two antiparallel β-sheets with 7 main strands, packing against each other, forming a β-sandwich. Compared to its homolog, Ndt80, MyRF has a smaller and less complex DBD lacking the helices and the big loops outside the core. Structural alignment reveals that MyRF DBD possess less interaction sites with DNA than Ndt80 and may bind only at the major groove of DNA. Moreover, the structure reveals a trimeric assembly, agreeing with the previous report that MyRF DBD functions as a trimer. The mutant that we designed based on the structure disturbed trimer formation, but didn’t affect the auto-cleavage reaction. It demonstrates that the activation of self-cleavage reaction of MyRF is independent of the presence of its N-terminal DBD homotrimer. The structure reported here will help to understand the molecular mechanism underlying the important roles of MyRF in myelin formation and development.

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“…coli [6, 10, 34–39]. On the contrary, information about structural-function relationships of enzymes from DA type 5 is scarce, with only two structures solved (BgaD-D from Bacillus circulans and BgaA from Streptococcus pneumoniae ) [30, 40] and no site-directed mutagenesis studies within the catalytic pocket.…”

Section: Resultsmentioning

confidence: 99%

“…This complex may be considered an analogue of the galactosyl covalent intermediate in the reaction mechanism, bound to the acceptor lactose. One of the residues interacting with this ligand is located within the GH2N domain, whereas the other 13 reside in the GH2C domain, including the catalytic triad formed by Glu 447 (acid base), Glu 532 (nucleophile) and Tyr 511 [40]. Most contacts would be established with the galactosyl moiety located deeper in the active site, which would be covalently linked to the enzyme in the reaction intermediate.…”

Section: Resultsmentioning

confidence: 99%

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Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2)

et al. 2016

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In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2). We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module) with two β-sandwich domains (non-catalytic) at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

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Crystal structure of β‐galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants

Cited by 46 publications

References 39 publications

Monobody-mediated alteration of enzyme specificity

Monobody-mediated alteration of enzyme specificity

Crystal structure of the DNA-binding domain of Myelin-gene Regulatory Factor

Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2)

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