Functional Characterization of the C-terminal Domain of the CytochromecMaturation Protein CcmE

The giant virus Mimivirus encodes an autonomous glycosylation system that is thought to be responsible for the formation of complex and unusual glycans composing the fibers surrounding its icosahedral capsid, including the dideoxyhexose viosamine. Previous studies have identified a gene cluster in the virus genome, encoding enzymes involved in nucleotide-sugar production and glycan formation, but the functional characterization of these enzymes and the full identification of the glycans found in viral fibers remain incomplete. Because viosamine is typically found in acylated forms, we suspected that one of the genes might encode an acyltransferase, providing directions to our functional annotations. Bioinformatic analyses indicated that the L142 protein contains an N-terminal acyltransferase domain and a predicted C-terminal glycosyltransferase. Sequence analysis of the structural model of the L142 N-terminal domain indicated significant homology with some characterized sugar acetyltransferases that modify the C-4 amino group in the bacillosamine or perosamine biosynthetic pathways. Using mass spectrometry and NMR analyses, we confirmed that the L142 N-terminal domain is a sugar acetyltransferase, catalyzing the transfer of an acetyl moiety from acetyl-CoA to the C-4 amino group of UDP-d-viosamine. The presence of acetylated viosamine in vivo has also been confirmed on the glycosylated viral fibers, using GC-MS and NMR. This study represents the first report of a virally encoded sugar acetyltransferase.

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Structural and functional characterization of the N-terminal domain of the yeast Mg2+channel Mrs2

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913011712 ◽

2013 ◽

Vol 69 (9) ◽

pp. 1653-1664 ◽

Cited By ~ 7

Author(s):

Muhammad Bashir Khan ◽

Gerhard Sponder ◽

Björn Sjöblom ◽

Soňa Svidová ◽

Rudolf J. Schweyen ◽

...

Keyword(s):

Functional Characterization ◽

Terminal Domain

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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.

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Structural and functional characterization of an Isd-type haem-degradation enzyme fromListeria monocytogenes

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713030794 ◽

2014 ◽

Vol 70 (3) ◽

pp. 615-626 ◽

Cited By ~ 13

Author(s):

Thao Duong ◽

Kwangsu Park ◽

Truc Kim ◽

Sung Wook Kang ◽

Myung Joon Hahn ◽

...

Keyword(s):

Crystal Structure ◽

Degradation Product ◽

Functional Characterization ◽

Human Pathogen ◽

Monooxygenase Activity ◽

Gram Positive Bacteria ◽

Functional Assays ◽

Terminal Domain ◽

Life Threatening

Bacterial pathogens have evolved diverse types of efficient machinery to acquire haem, the most abundant source of iron in the human body, and degrade it for the utilization of iron. Gram-positive bacteria commonly encode IsdG-family proteins as haem-degrading monooxygenases.Listeria monocytogenesis predicted to possess an IsdG-type protein (Lmo2213), but the residues involved in haem monooxygenase activity are not well conserved and there is an extra N-terminal domain in Lmo2213. Therefore, its function and mechanism of action cannot be predicted. In this study, the crystal structure of Lmo2213 was determined at 1.75 Å resolution and its haem-binding and haem-degradation activities were confirmed. Structure-based mutational and functional assays of this protein, designated as an Isd-typeL. monocytogeneshaem-degrading enzyme (Isd-LmHde), identified that Glu71, Tyr87 and Trp129 play important roles in haem degradation and that the N-terminal domain is also critical for its haem-degrading activity. The haem-degradation product of Isd-LmHde is verified to be biliverdin, which is also known to be the degradation product of other bacterial haem oxygenases. This study, the first structural and functional report of the haem-degradation system inL. monocytogenes, sheds light on the concealed haem-utilization system in this life-threatening human pathogen.

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Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli

PLoS ONE ◽

10.1371/journal.pone.0187051 ◽

2017 ◽

Vol 12 (10) ◽

pp. e0187051 ◽

Cited By ~ 5

Author(s):

Christine Belloir ◽

Jimmy Savistchenko ◽

Fabrice Neiers ◽

Andrew J. Taylor ◽

Scott McGrane ◽

...

Keyword(s):

Escherichia Coli ◽

Functional Characterization ◽

Taste Receptor ◽

Umami Taste ◽

Terminal Domain

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Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly

Life Science Alliance ◽

10.26508/lsa.201900630 ◽

2020 ◽

Vol 3 (11) ◽

pp. e201900630

Author(s):

María Ascensión Villar-Fernández ◽

Richard Cardoso da Silva ◽

Magdalena Firlej ◽

Dongqing Pan ◽

Elisabeth Weir ◽

...

Keyword(s):

Biochemical Analysis ◽

Budding Yeast ◽

Functional Characterization ◽

Efficient Removal ◽

Terminal Domain ◽

Origins Of Replication ◽

Mcm Helicase ◽

A Subunit ◽

Aaa Protein

Pch2 is a meiosis-specific AAA+ protein that controls several important chromosomal processes. We previously demonstrated that Orc1, a subunit of the ORC, functionally interacts with budding yeast Pch2. The ORC (Orc1-6) AAA+ complex loads the AAA+ MCM helicase to origins of replication, but whether and how ORC collaborates with Pch2 remains unclear. Here, we show that a Pch2 hexamer directly associates with ORC during the meiotic G2/prophase. Biochemical analysis suggests that Pch2 uses its non-enzymatic NH2-terminal domain and AAA+ core and likely engages the interface of ORC that also binds to Cdc6, a factor crucial for ORC-MCM binding. Canonical ORC function requires association with origins, but we show here that despite causing efficient removal of Orc1 from origins, nuclear depletion of Orc2 and Orc5 does not trigger Pch2/Orc1-like meiotic phenotypes. This suggests that the function for Orc1/Pch2 in meiosis can be executed without efficient association of ORC with origins of replication. In conclusion, we uncover distinct functionalities for Orc1/ORC that drive the establishment of a non-canonical, meiosis-specific AAA+ assembly with Pch2.

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