Structural and biochemical characterization of bifunctional XynA

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

Download Full-text

Structure-function relationship of bifunctional XynA

10.22541/au.160970138.89944623/v1 ◽

2021 ◽

Author(s):

Wei Xie ◽

Qi Yu ◽

Ruiqin Zhang ◽

Yun Liu ◽

Ruoting Cao ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Catalytic Mechanism ◽

Cellulase Activity ◽

Cost Savings ◽

Site Directed Mutagenesis ◽

Enzyme Activity Assay ◽

Great Cost ◽

Terminal Domain ◽

Gh10 Family

Xylan and cellulose are the two major constituents in numerous types of lignocellulose. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. was found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. XynA has a longer N-terminal and C-terminal than most other GH10 family enzymes. The important thing is that the activity of our N-terminal truncated XynA_ΔN37 is significantly improved. And we found that the C-terminus is crucial to protein expression in solution. Protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with double mutant E182A/E280A. Molecular docking studies of XynA with xylohexaose and cellohexaose, together with site-directed mutagenesis and enzyme activity assay, demonstrate that Gln250 and His252 are indispensable to bifunctional activity. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

Download Full-text

Design and Synthesis of N-phenyl Phthalimides as Potent Protoporphyrinogen Oxidase Inhibitors

Molecules ◽

10.3390/molecules24234363 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4363

Author(s):

Wei Gao ◽

Xiaotian Li ◽

Da Ren ◽

Susu Sun ◽

Jingqian Huo ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Molecular Docking ◽

Molecular Design ◽

Design Strategy ◽

Docking Studies ◽

Activity Assay ◽

Enzyme Activity Assay ◽

Protoporphyrinogen Oxidase ◽

Design And Synthesis

Protoporphyrinogen oxidase (PPO) has been identified as one of the most promising targets for herbicide discovery. A series of novel phthalimide derivatives were designed by molecular docking studies targeting the crystal structure of mitochondrial PPO from tobacco (mtPPO, PDB: 1SEZ) by using Flumioxazin as a lead, after which the derivatives were synthesized and characterized, and their herbicidal activities were subsequently evaluated. The herbicidal bioassay results showed that compounds such as 3a (2-(4-bromo-2,6-difluorophenyl) isoindoline-1,3-dione), 3d (methyl 2-(4-chloro-1,3-dioxoisoindolin-2-yl)-5-fluorobenzoate), 3g (4-chloro-2-(5-methylisoxazol-3-yl) isoindoline-1,3-dione), 3j (4-chloro-2-(thiophen-2-ylmethyl) isoindoline-1,3-dione) and 3r (2-(4-bromo-2,6-difluorophenyl)-4-fluoroisoindoline-1,3-dione) had good herbicidal activities; among them, 3a showed excellent herbicidal efficacy against A. retroflexus and B. campestris via the small cup method and via pre-emergence and post-emergence spray treatments. The efficacy was comparable to that of the commercial herbicides Flumioxazin, Atrazine, and Chlortoluron. Further, the enzyme activity assay results suggest that the mode of action of compound 3a involves the inhibition of the PPO enzyme, and 3a showed better inhibitory activity against PPO than did Flumioxazin. These results indicate that our molecular design strategy contributes to the development of novel promising PPO inhibitors.

Download Full-text

Biochemical Characterization of Laboratory Mutants of Extended-Spectrum β-Lactamase TEM-60

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.9.3579-3582.2004 ◽

2004 ◽

Vol 48 (9) ◽

pp. 3579-3582 ◽

Cited By ~ 6

Author(s):

Bibiana Caporale ◽

Nicola Franceschini ◽

Mariagrazia Perilli ◽

Bernardetta Segatore ◽

Gian Maria Rossolini ◽

...

Keyword(s):

Enzyme Activity ◽

Kinetic Parameters ◽

Biochemical Characterization ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Extended Spectrum

ABSTRACT Three mutants of the extended-spectrum β-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

Download Full-text

Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase fromRhodococcus erythropolisSQ1 Explain Its Catalytic Mechanism

Journal of Biological Chemistry ◽

10.1074/jbc.m113.522771 ◽

2013 ◽

Vol 288 (49) ◽

pp. 35559-35568 ◽

Cited By ~ 26

Author(s):

Ali Rohman ◽

Niels van Oosterwijk ◽

Andy-Mark W. H. Thunnissen ◽

Bauke W. Dijkstra

Keyword(s):

Crystal Structure ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

Download Full-text

Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

Nucleic Acids Research ◽

10.1093/nar/gkab799 ◽

2021 ◽

Author(s):

Keisuke Oki ◽

Mariko Nagata ◽

Takeshi Yamagami ◽

Tomoyuki Numata ◽

Sonoko Ishino ◽

...

Keyword(s):

Crystal Structure ◽

Dna Polymerase ◽

Direct Interaction ◽

Crystal Structure Analysis ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Functional Connection ◽

Terminal Domain ◽

The Family ◽

Lagging Strand

Abstract Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

Download Full-text

Structural insights into SETD3-mediated histidine methylation on β-actin

10.1101/476705 ◽

2018 ◽

Author(s):

Qiong Guo ◽

Shanhui Liao ◽

Sebastian Kwiatkowski ◽

Weronika Tomaka ◽

Huijuan Yu ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Enzyme Activity ◽

Small Molecule ◽

Conformational Changes ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Domain Protein ◽

First Time ◽

Structural Insights

SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Recently, we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of β-actin by SETD3 is highly sequence specific, and both SETD3 and β-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the data show for the first time a catalytic mechanism of SETD3-mediated histidine methylation in β-actin, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.

Download Full-text

Classification of a VUS in MLH1 using a combination of family segregation studies and protein biochemistry.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.4_suppl.342 ◽

2013 ◽

Vol 31 (4_suppl) ◽

pp. 342-342

Author(s):

Kevin M. Zbuk ◽

Kathleen Bell ◽

Anna Zhou ◽

Alba Guarne ◽

Melyssa Aronson ◽

...

Keyword(s):

Crystal Structure ◽

Missense Variant ◽

Site Directed Mutagenesis ◽

Multiple Sources ◽

Dimerization Domain ◽

Protein Biochemistry ◽

Dna Mismatch ◽

Terminal Domain ◽

Amsterdam Criteria

342 Background: The identification of variants of unknown clinical significance (VUS) presents a challenge for diagnostic laboratories and clinicians working with families with Lynch syndrome (LS). Although a variety of strategies exist to attempt clarification, data from multiple sources is often required before a VUS is re-classified to pathogenic or benign. Methods: We report on the investigation of 2 families with a variant in the MMR gene MLH1, c.2038T>C (p.Cys680Arg). In addition to segregation analysis, and review of published literature, the c-terminal domain of MLH1was cloned, the p.Cys680Arg variant was generated using site-directed mutagenesis and protein expression was induced. In addition, this point mutation was modelled onto the crystal structure of the dimerization domain of MLH1. Results: Both families fit Amsterdam criteria for a diagnosis of LS. All available tumours exhibited MLH1 deficiency and microsatellite instability and all living affected individuals available were positive for MLH1 p.Cys680Arg variant. Protein studies confirmed that p.Cys680 resides in a tight hydrophobic cavity and the p.Cys680Arg mutation disrupts the folding of the C-terminal domain of MLH1. In two families this variant segregated with disease in a total of 12 individuals (>10 meiosis and >1000:1 odds in favor of linkage) and 3 had an MLH1 deficient tumour, supporting a pathogenic role for this variant. Inspection of the crystal structure of MLH1 suggested that the Cys680Arg mutation should severely alter the folding of the dimerization domain. As opposed to the dimerization domain of MLH1 that can be readily produced recombinantly and is stable in solution, the same domain of MLH1 encoding the Cys680Arg mutation was expressed as inclusion bodies, confirming that this mutation causes misfolding of the dimerization domain of MLH1. Conclusions: Our data confirms that the p.Cys680Arg mutation destabilizes the dimerization domain of MLH1, and presumably the DNA mismatch repair proficiency of individuals carrying this mutation. Based on this evidence and family history data, the MLH1 C680R missense variant is classified as pathogenic. This approach may prove useful in the classification of other variants.

Download Full-text

Functional characterization of heat-shock protein 90 fromOryza sativaand crystal structure of its N-terminal domain

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15006639 ◽

2015 ◽

Vol 71 (6) ◽

pp. 688-696 ◽

Cited By ~ 1

Author(s):

Swetha Raman ◽

Kaza Suguna

Keyword(s):

Crystal Structure ◽

Heat Shock ◽

Heat Shock Protein ◽

Biochemical Characterization ◽

Functional Characterization ◽

Cell Signalling ◽

Physiological Role ◽

Heat Shock Protein 90 ◽

Terminal Domain

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 fromDictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

Download Full-text