Crystal structure of Bombyx mori nucleopolyhedrovirus ORF75 reveals a pseudo-dimer of thiol oxidase domains with a putative substrate-binding pocket

Bombyx mori nucleopolyhedrovirus (BmNPV) triggers the global shutdown of host silkworm gene expression and protein synthesis approximately 12–18 h post-infection. Genome sequence analysis suggests that BmNPV ORF75 could be a flavin adenine dinucleotide (FAD)-linked thiol oxidase essential for virion assembly and virus propagation. Here, we report the crystal structure of BmNPV ORF75 at 2.1 Å (0.21 nm). The structure of BmNPV ORF75 resembles that of the thiol oxidase domain of human quiescin thiol oxidase (QSOX), displaying a pseudo-dimer of canonical and non-canonical thiol oxidase domains. However, BmNPV ORF75 is further dimerized by its C-terminal canonical thiol oxidase domain. Within the unique quaternary structural arrangement, the FAD-binding pocket and the characteristic CXXC motif from each monomer is 35 Å (3.5 nm) away from that of its corresponding molecule, which suggests that BmNPV ORF75 might adopt a deviant mechanism from that of QSOX to catalyse disulfide bond formation. Our thiol oxidase activity assay on the point mutations of the conserved residues participating in FAD recognition reveals an aromatic cage next to the FAD isoalloxazine moiety for substrate binding. These data suggest that the thiol oxidase activity of BmNPV ORF75 could be critical to catalyse the formation of the disulfide bonds of certain BmNPV proteins essential for BmNPV virion assembly.

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Identification of the site of oxidase substrate binding inScytalidium thermophilumcatalase

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318010628 ◽

2018 ◽

Vol 74 (10) ◽

pp. 979-985 ◽

Cited By ~ 2

Author(s):

Yonca Yuzugullu Karakus ◽

Gunce Goc ◽

Sinem Balci ◽

Briony A. Yorke ◽

Chi H. Trinh ◽

...

Keyword(s):

Crystal Structure ◽

Kinetic Analysis ◽

Binding Site ◽

Active Site ◽

Oxidase Activity ◽

Substrate Binding ◽

Binding Pocket ◽

Scytalidium Thermophilum ◽

Lateral Channel

The catalase fromScytalidium thermophilumis a homotetramer containing a hemedin each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95 Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.

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Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

Nature Communications ◽

10.1038/s41467-020-20675-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yufei Han ◽

Qian Zhuang ◽

Bo Sun ◽

Wenping Lv ◽

Sheng Wang ◽

...

Keyword(s):

Crystal Structure ◽

Biochemical Characterization ◽

Substrate Binding ◽

Binding Pocket ◽

Substrate Binding Pocket ◽

Transmembrane Segments ◽

Therapeutic Molecules ◽

Binding Cavity ◽

Immune System Regulation ◽

Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

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Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803685115 ◽

2018 ◽

Vol 115 (17) ◽

pp. E3932-E3939 ◽

Cited By ~ 4

Author(s):

Maria C. Garcia-Guerrero ◽

Javier Garcia-Pardo ◽

Esther Berenguer ◽

Roberto Fernandez-Alvarez ◽

Gifty B. Barfi ◽

...

Keyword(s):

Crystal Structure ◽

Specific Activity ◽

Dominant Role ◽

Substrate Binding ◽

Digestive Enzyme ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Enzyme Specificity ◽

Substrate Binding Pocket ◽

Acidic Residues

Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor. The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at “canonical” position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1′ substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.

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The structure of the hexameric atrazine chlorohydrolase AtzA

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715000619 ◽

2015 ◽

Vol 71 (3) ◽

pp. 710-720 ◽

Cited By ~ 14

Author(s):

T. S. Peat ◽

J. Newman ◽

S. Balotra ◽

D. Lucent ◽

A. C. Warden ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Binding ◽

Physiological Role ◽

Binding Pocket ◽

Soil Organism ◽

Substrate Binding Pocket ◽

Herbicide Atrazine ◽

Hexameric Form ◽

Considerable Strain

Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organismPseudomonassp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role (i.e.atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit.

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The crystal structure of Escherichia coli spermidine synthase SpeE reveals a unique substrate-binding pocket

Journal of Structural Biology ◽

10.1016/j.jsb.2009.12.024 ◽

2010 ◽

Vol 169 (3) ◽

pp. 277-285 ◽

Cited By ~ 12

Author(s):

Xingding Zhou ◽

Teck Khiang Chua ◽

Karolina L. Tkaczuk ◽

Janusz M. Bujnicki ◽

J. Sivaraman

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Substrate Binding ◽

Binding Pocket ◽

Spermidine Synthase ◽

Substrate Binding Pocket

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Crystal structure of the dimethylsulfide monooxygenase DmoA from Hyphomicrobium sulfonivorans

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18015844 ◽

2018 ◽

Vol 74 (12) ◽

pp. 781-786 ◽

Cited By ~ 1

Author(s):

Hai-Yan Cao ◽

Peng Wang ◽

Ming Peng ◽

Xuan Shao ◽

Xiu-Lan Chen ◽

...

Keyword(s):

Crystal Structure ◽

Sequence Similarity ◽

Substrate Binding ◽

Detailed Comparison ◽

Binding Pocket ◽

Flavin Mononucleotide ◽

Substrate Binding Pocket ◽

High Sequence Similarity ◽

Tim Barrel

DmoA is a monooxygenase which uses dioxygen (O2) and reduced flavin mononucleotide (FMNH2) to catalyze the oxidation of dimethylsulfide (DMS). Although it has been characterized, the structure of DmoA remains unknown. Here, the crystal structure of DmoA was determined to a resolution of 2.28 Å and was compared with those of its homologues LadA and BdsA. The results showed that their overall structures are similar: they all share a conserved TIM-barrel fold which is composed of eight α-helices and eight β-strands. In addition, they all have five additional insertions. Detailed comparison showed that the structures have notable differences despite their high sequence similarity. The substrate-binding pocket of DmoA is smaller compared with those of LadA and BdsA.

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