Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate

The L,D-carboxypeptidase DacB plays a key role in the remodelling ofStreptococcus pneumoniaepeptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Å resolution crystal structure of DacB fromS. pneumoniaeTIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB fromS. pneumoniaeD39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, withKmandkcatvalues of 2.84 ± 0.37 mMand 91.49 ± 0.05 s−1, respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.

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β-Amyrin biosynthesis: catalytic mechanism and substrate recognition

Organic & Biomolecular Chemistry ◽

10.1039/c7ob00238f ◽

2017 ◽

Vol 15 (14) ◽

pp. 2869-2891 ◽

Cited By ~ 21

Author(s):

Tsutomu Hoshino

Keyword(s):

Catalytic Mechanism ◽

Substrate Recognition ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

The Past ◽

Substrate Analog ◽

Analog Experiments

In the past five years, there have been remarkable advances in the study of β-amyrin synthase. This review outlines the catalytic mechanism and substrate recognition in β-amyrin biosynthesis, which have been attained by the site-directed mutagenesis and substrate analog experiments.

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Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713034275 ◽

2014 ◽

Vol 70 (4) ◽

pp. 1166-1172 ◽

Cited By ~ 5

Author(s):

Michael E. Webb ◽

Briony A. Yorke ◽

Tom Kershaw ◽

Sarah Lovelock ◽

Carina M. C. Lobley ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Site Directed Mutagenesis ◽

Intramolecular Rearrangement ◽

Directed Mutagenesis ◽

Vitamin B ◽

Biosynthesis Pathway ◽

Serine Residue ◽

Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

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Crystal Structure and Site-directed Mutagenesis ofBacillus maceransEndo-1,31,4--glucanase

Journal of Biological Chemistry ◽

10.1074/jbc.270.7.3081 ◽

1995 ◽

Vol 270 (7) ◽

pp. 3081-3088 ◽

Cited By ~ 76

Author(s):

Michael Hahn ◽

Ole Olsen ◽

Oliver Politz ◽

Rainer Borriss ◽

Udo Heinemann

Keyword(s):

Crystal Structure ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis

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Structural and biochemical characterization of bifunctional XynA

10.1101/2020.10.20.348094 ◽

2020 ◽

Author(s):

Wei Xie ◽

Qi Yu ◽

Yun Liu ◽

Ruoting Cao ◽

Ruiqing Zhang ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Catalytic Mechanism ◽

Biochemical Characterization ◽

Cellulase Activity ◽

Cost Savings ◽

Site Directed Mutagenesis ◽

Enzyme Activity Assay ◽

Great Cost ◽

Terminal Domain

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.

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Crystal Structure and Site-directed Mutagenesis of Enzymatic Components from Clostridium perfringens Iota-toxin

Journal of Molecular Biology ◽

10.1016/s0022-2836(02)01247-0 ◽

2003 ◽

Vol 325 (3) ◽

pp. 471-483 ◽

Cited By ~ 78

Author(s):

Hideaki Tsuge ◽

Masahiro Nagahama ◽

Hiroyuki Nishimura ◽

Junzo Hisatsune ◽

Yoshihiko Sakaguchi ◽

...

Keyword(s):

Crystal Structure ◽

Clostridium Perfringens ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Clostridium Perfringens Iota Toxin

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Conformational communication mediates the reset step in t6A biosynthesis

Nucleic Acids Research ◽

10.1093/nar/gkz439 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6551-6567 ◽

Cited By ~ 6

Author(s):

Amit Luthra ◽

Naduni Paranagama ◽

William Swinehart ◽

Susan Bayooz ◽

Phuc Phan ◽

...

Keyword(s):

Crystal Structure ◽

Molecular Docking ◽

Atpase Activity ◽

Atp Hydrolysis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Translational Fidelity ◽

Substrate Analog ◽

Binding Cavity ◽

Saxs Analysis

Abstract The universally conserved N6-threonylcarbamoyladenosine (t6A) modification of tRNA is essential for translational fidelity. In bacteria, t6A biosynthesis starts with the TsaC/TsaC2-catalyzed synthesis of the intermediate threonylcarbamoyl adenylate (TC–AMP), followed by transfer of the threonylcarbamoyl (TC) moiety to adenine-37 of tRNA by the TC-transfer complex comprised of TsaB, TsaD and TsaE subunits and possessing an ATPase activity required for multi-turnover of the t6A cycle. We report a 2.5-Å crystal structure of the T. maritima TC-transfer complex (TmTsaB2D2E2) bound to Mg2+-ATP in the ATPase site, and substrate analog carboxy-AMP in the TC-transfer site. Site directed mutagenesis results show that residues in the conserved Switch I and Switch II motifs of TsaE mediate the ATP hydrolysis-driven reactivation/reset step of the t6A cycle. Further, SAXS analysis of the TmTsaB2D2-tRNA complex in solution reveals bound tRNA lodged in the TsaE binding cavity, confirming our previous biochemical data. Based on the crystal structure and molecular docking of TC–AMP and adenine-37 in the TC-transfer site, we propose a model for the mechanism of TC transfer by this universal biosynthetic system.

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