Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity

Pathogenic bacteria have acquired a vast array of eukaryotic-like proteins via intimate interaction with host cells. Bacterial effector proteins that function as ubiquitin ligases and deubiquitinases (DUBs) are remarkable examples of such molecular mimicry. LotA, a Legionella pneumophila effector, belongs to the ovarian tumor (OTU) superfamily, which regulates diverse ubiquitin signals by their DUB activities. LotA harbors two OTU domains that have distinct reactivities; the first one is responsible for the cleavage of the K6-linked ubiquitin chain, and the second one shows an uncommon preference for long chains of ubiquitin. Here, we report the crystal structure of a middle domain of LotA (LotA M ), which contains the second OTU domain. LotA M consists of two distinct subdomains, a catalytic domain having high structural similarity with human OTU DUBs and an extended helical lobe (EHL) domain, which is characteristically conserved only in Legionella OTU DUBs. The docking simulation of LotA M with ubiquitin suggested that hydrophobic and electrostatic interactions between the EHL of LotA M and the C-terminal region of ubiquitin are crucial for the binding of ubiquitin to LotA M . The structure-based mutagenesis demonstrated that the acidic residue in the characteristic short helical segment termed the ‘helical arm’ is essential for the enzymatic activity of LotA M . The EHL domain of the three Legionella OTU DUBs, LotA, LotB, and LotC, share the ‘helical arm’ structure, suggesting that the EHL domain defines the Lot-OTUs as a unique class of DUBs. Importance To successfully colonize, some pathogenic bacteria hijack the host ubiquitin system. Legionella OTU-like-DUBs (Lot-DUBs) are novel bacterial deubiquitinases found in effector proteins of L. pneumophila . LotA is a member of Lot-DUBs and has two OTU domains (OTU1 and OTU2). We determined the structure of a middle fragment of LotA (LotA M ), which includes OTU2. LotA M consists of the conserved catalytic domain and the Legionella OTUs-specific EHL domain. The docking simulation with ubiquitin and the mutational analysis suggested that the acidic surface in the EHL is essential for enzymatic activity. The structure of the EHL differs from those of other Lot-DUBs, suggesting that the variation of the EHL is related to the variable cleaving specificity of each DUB.

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Structural basis for topological regulation of Tn3 resolvase

10.1101/2021.12.07.471667 ◽

2021 ◽

Author(s):

Sherwin P Montaño ◽

Sally-J Rowland ◽

James R. Fuller ◽

Mary E. Burke ◽

Alasdair I. MacDonald ◽

...

Keyword(s):

Experimental Data ◽

Antibiotic Resistance ◽

Synthetic Biology ◽

Enzymatic Activity ◽

Dna Topology ◽

Structural Basis ◽

Detailed Model ◽

Site Specific ◽

Site Specific Recombination ◽

Dna Complex

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely-related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

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Design, Synthesis, and Preliminary Bioactivity Evaluation of 2,7-Substituted Carbazole Derivatives as Potent Autotaxin Inhibitors and Antitumor Agents†

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180830161821 ◽

2019 ◽

Vol 19 (2) ◽

pp. 256-264

Author(s):

Wenming Wang ◽

Fengmei Zhao ◽

Yarui Zhao ◽

Weiwei Pan ◽

Pengcheng Cao ◽

...

Keyword(s):

Molecular Docking ◽

Enzymatic Activity ◽

Cancer Progression ◽

Anticancer Agents ◽

Antitumor Agents ◽

Hydrophobic Interactions ◽

Human Cancer ◽

Structural Basis ◽

Human Carcinoma ◽

Carbazole Derivatives

Background: Autotaxin-LPA signaling has been implicated in cancer progression, and targeted for the discovery of cancer therapeutic agents. Objective: Potential ATX inhibitors were synthesized to develop novel leading compounds and effective anticancer agents. Methods: The present work designs and synthesizes a series of 2,7-subsitituted carbazole derivatives with different terminal groups R [R = -Cl (I), -COOH (II), -B(OH)2 (III), or -PO(OH)2 (I-IV)]. The inhibition of these compounds on the enzymatic activity of ATX was measured using FS-3 and Bis-pNpp as substrates, and the cytotoxicity of these compounds was evaluated using SW620, SW480, PANC-1, and SKOV-3 human carcinoma cells. Furthermore, the binding of leading compound with ATX was analyzed by molecular docking. Results: Compound III was shown to be a promising antitumor candidate by demonstrating both good inhibition of ATX enzymatic activity and high cytotoxicity against human cancer cell lines. Molecular docking study shows that compound III is located in a pocket, which mainly comprises amino acids 209 to 316 in domain 2 of ATX, and binds with these residues of ATX through van der Waals, conventional hydrogen bonds, and hydrophobic interactions. Conclusion: Compound III with the terminal group R = -B(OH)2 has the most potent inhibitory effect with the greatest cytotoxicity to cancer cells. Moreover, the docking model provides a structural basis for the future optimization of promising antitumor compounds.

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Structural characterization of the isoenzymatic forms of human myeloperoxidase: evaluation of the iron-containing prosthetic group

Blood ◽

10.1182/blood.v75.1.238.bloodjournal751238 ◽

1990 ◽

Vol 75 (1) ◽

pp. 238-241

Author(s):

J Wright ◽

N Bastian ◽

TA Davis ◽

C Zuo ◽

S Yoshimoto ◽

...

Keyword(s):

Enzymatic Activity ◽

Exchange Chromatography ◽

Structural Basis ◽

Human Neutrophils ◽

Prosthetic Group ◽

Paramagnetic Resonance ◽

Structural Identity ◽

Significant Difference ◽

Cellular Compartmentalization

Myeloperoxidase (MPO) from human neutrophils has been purified and found to exist in three isoenzymatic forms, resolved by ion exchange chromatography. In addition to differences in subunit size and cellular compartmentalization of the isoenzymes, differences have been reported in their activity and susceptibility to inhibition. The structural basis of these isoenzymes is unclear; we attempted to further define their functional characteristics and structural identity. First, we measured respective enzymatic activity using a panel of substrates; MPO I was found to have lower activity with some substrates (pyrogallol, guaiacol, potassium iodide [KI]), but similar activity to the other isoenzymes with 4-aminoantipyrine. These studies confirm that MPO I is enzymatically distinct from MPO II and MPO III. Next, we examined the structural basis of these differences by evaluating the iron-containing prosthetic group in each form using electron paramagnetic resonance (EPR) and determination of the pyridine hemochrome. No significant difference between the isoenzymes was noted in these parameters, suggesting that the prosthetic group is the same in each protein. The cause for any difference in enzymatic activity must lie then in variations extrinsic to the heme, and based on previous studies of the gene and protein analysis, the posttranslational modification of MPO must account for these isoenzymatic species.

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Structural basis of the significant calmodulin-induced increase in the enzymatic activity of secreted phospholipases A2

Protein Engineering Design and Selection ◽

10.1093/protein/gzq019 ◽

2010 ◽

Vol 23 (6) ◽

pp. 479-487 ◽

Cited By ~ 10

Author(s):

Lidija Kovačič ◽

Marko Novinec ◽

Toni Petan ◽

Igor Križaj

Keyword(s):

Enzymatic Activity ◽

Structural Basis ◽

Phospholipases A2

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Structural characterization of the isoenzymatic forms of human myeloperoxidase: evaluation of the iron-containing prosthetic group

Blood ◽

10.1182/blood.v75.1.238.238 ◽

1990 ◽

Vol 75 (1) ◽

pp. 238-241 ◽

Cited By ~ 6

Author(s):

J Wright ◽

N Bastian ◽

TA Davis ◽

C Zuo ◽

S Yoshimoto ◽

...

Keyword(s):

Enzymatic Activity ◽

Exchange Chromatography ◽

Structural Basis ◽

Human Neutrophils ◽

Prosthetic Group ◽

Paramagnetic Resonance ◽

Structural Identity ◽

Significant Difference ◽

Cellular Compartmentalization

Abstract Myeloperoxidase (MPO) from human neutrophils has been purified and found to exist in three isoenzymatic forms, resolved by ion exchange chromatography. In addition to differences in subunit size and cellular compartmentalization of the isoenzymes, differences have been reported in their activity and susceptibility to inhibition. The structural basis of these isoenzymes is unclear; we attempted to further define their functional characteristics and structural identity. First, we measured respective enzymatic activity using a panel of substrates; MPO I was found to have lower activity with some substrates (pyrogallol, guaiacol, potassium iodide [KI]), but similar activity to the other isoenzymes with 4-aminoantipyrine. These studies confirm that MPO I is enzymatically distinct from MPO II and MPO III. Next, we examined the structural basis of these differences by evaluating the iron-containing prosthetic group in each form using electron paramagnetic resonance (EPR) and determination of the pyridine hemochrome. No significant difference between the isoenzymes was noted in these parameters, suggesting that the prosthetic group is the same in each protein. The cause for any difference in enzymatic activity must lie then in variations extrinsic to the heme, and based on previous studies of the gene and protein analysis, the posttranslational modification of MPO must account for these isoenzymatic species.

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