Crystal Structures of Putative Flavin Dependent Monooxygenase from Alicyclobacillus Acidocaldarius

Flavin dependent monooxygenases catalyze various reactions to play a key role in biological processes, such as catabolism, detoxification, and biosynthesis. Group D flavin dependent monooxygenases are enzymes with an Acyl-CoA dehydrogenase (ACAD) fold and use Flavin adenine dinucleotide (FAD) or Flavin mononucleotide (FMN) as a cofactor. In this research, crystal structures of Alicyclobacillus acidocaldarius protein formerly annotated as an ACAD were determined in Apo and FAD bound state. Although our structure showed high structural similarity to other ACADs, close comparison of substrate binding pocket and phylogenetic analysis showed that this protein is more closely related to other bacterial group D flavin dependent monooxygenases, such as DszC (sulfoxidase) and DnmZ and Kijd3 (nitrososynthases).

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Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0905270106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15616-15621 ◽

Cited By ~ 30

Author(s):

Masataka Umitsu ◽

Hiroshi Nishimasu ◽

Akiko Noma ◽

Tsutomu Suzuki ◽

Ryuichiro Ishitani ◽

...

Keyword(s):

Crystal Structures ◽

Binding Pocket ◽

Structural Basis ◽

Binding Modes ◽

Substrate Binding Pocket ◽

Methyl Group ◽

Group Transfer ◽

Canonical Class ◽

Functional Analyses

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.

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Crystal structures of the disulfide reductase DsbM from Pseudomonas aeruginosa

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316013024 ◽

2016 ◽

Vol 72 (10) ◽

pp. 1100-1109 ◽

Cited By ~ 1

Author(s):

Inseong Jo ◽

Nohra Park ◽

In-Young Chung ◽

You-Hee Cho ◽

Nam-Chul Ha

Keyword(s):

Pseudomonas Aeruginosa ◽

Crystal Structures ◽

Structural Similarity ◽

Binding Pocket ◽

Glutathione S Transferase ◽

Redox Sensor ◽

Substrate Peptide ◽

Reactive Oxygen Species Stress ◽

Lid Domain ◽

Substrate Proteins

In bacteria, many Dsb-family proteins play diverse roles in the conversion between the oxidized and reduced states of cysteine residues of substrate proteins. Most Dsb enzymes catalyze disulfide formation in periplasmic or secreted substrate proteins. Recently, a DsbM protein has been found in a Gram-negative bacterium, and was characterized as a cytosolic Dsb member with the conserved CXXC motif on the basis of sequence homology to the Dsb-family proteins. The protein was implicated in the reduction of the cytoplasmic redox-sensor protein OxyR in Pseudomonas aeruginosa. Here, crystal structures of DsbM from P. aeruginosa are presented, revealing that it consists of a modified thioredoxin domain containing the CXXC motif and a lid domain surrounding the CXXC motif. In a glutathione-linked structure, a glutathione molecule is linked to the CXXC motif of DsbM and is bound in an elongated cavity region in the thioredoxin domain, which is also suited for substrate peptide binding. A striking structural similarity to a human glutathione S-transferase was found in the glutathione-binding pocket. Further, biochemical evidence is presented suggesting that DsbM is directly involved in the reduction of the disulfide of Cys199 and Cys208 in OxyR, resulting in the acceleration of OxyR reduction in the absence of reactive oxygen species stress. These findings may help to expand the understanding of the diverse roles of redox-related proteins that contain the CXXC motif.

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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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Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation–π interaction

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321008937 ◽

2021 ◽

Vol 77 (10) ◽

Author(s):

F. Helfrich ◽

Axel J. Scheidig

Keyword(s):

Pseudomonas Aeruginosa ◽

Crystal Structures ◽

Active Site ◽

Structural Similarity ◽

Opportunistic Pathogen ◽

Binding Pocket ◽

Polyamine Metabolism ◽

Tryptophan Residue ◽

Salt Bridges ◽

Blastochloris Viridis

Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (`ionic slide') and near the active site (`inner amino site') to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation–π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.

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Structural Insights into Azole-based Inhibitors of Heme Oxygenase-1: Development of Selective Compounds for Therapeutic Applications

Current Medicinal Chemistry ◽

10.2174/0929867325666180606082512 ◽

2019 ◽

Vol 25 (42) ◽

pp. 5803-5821 ◽

Cited By ~ 3

Author(s):

Mona N. Rahman ◽

Dragic Vukomanovic ◽

Jason Z. Vlahakis ◽

Walter A. Szarek ◽

Kanji Nakatsu ◽

...

Keyword(s):

Heme Oxygenase ◽

Competitive Inhibition ◽

Cytochromes P450 ◽

Structural Similarity ◽

Binding Pocket ◽

Initial Study ◽

Structural Basis ◽

Heme Oxygenase 1 ◽

Design Strategies ◽

Inhibitor Binding

The development of isozyme-selective heme oxygenase (HO) inhibitors promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties with a role in several disease states; thus, it is an enticing therapeutic target. Historically, the metalloporphyrins have been used as competitive HO inhibitors based on their structural similarity to the substrate, heme. However, heme’s important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), results in non-selectivity being an unfortunate side effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort over a decade ago to develop novel compounds as potent, selective inhibitors of HO. The result was the creation of the first generation of non-porphyrin based, non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated and provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. Notably, HO-1 inhibitors are of particular interest for the treatment of hyperbilirubinemia and certain types of cancer. Key features based on this initial study have already been used by others to discover additional potential HO-1 inhibitors. Moreover, studies have begun to use selected compounds and determine their effects in some disease models.

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Microwave Imaging of Breast Skin Utilizing Elliptical UWB Antenna and Reverse Problems Algorithm

Micromachines ◽

10.3390/mi12060647 ◽

2021 ◽

Vol 12 (6) ◽

pp. 647

Author(s):

Sameer Alani ◽

Zahriladha Zakaria ◽

Tale Saeidi ◽

Asmala Ahmad ◽

Muhammad Ali Imran ◽

...

Keyword(s):

Skin Cancer ◽

Imaging System ◽

Signal Transmission ◽

Similarity Index ◽

Structural Similarity ◽

Microwave Imaging ◽

Ultra Wideband ◽

Uwb Antenna ◽

System A ◽

High Structural Similarity

Skin cancer is one of the most widespread and fast growing of all kinds of cancer since it affects the human body easily due to exposure to the Sun’s rays. Microwave imaging has shown better outcomes with higher resolution, faster processing time, mobility, and less cutter and artifact effects. A miniaturized elliptical ultra-wideband (UWB) antenna and its semi-spherical array arrangement were used for signal transmission and reception from the defected locations in the breast skin. Several conditions such as various arrays of three, six, and nine antenna elements, smaller tumor, multi-tumors, and skin on a larger breast sample of 30 cm were considered. To assess the ability of the system, a breast shape container with a diameter of 130 mm and height of 60 mm was 3D printed and then filled with fabricated skin and breast fat to perform the experimental investigation. An improved modified time-reversal algorithm (IMTR) was used to recreate 2D images of tumors with the smallest radius of 1.75 mm in any location within the breast skin. The reconstructed images using both simulated and experimental data verified that the system can be a reliable imaging system for skin cancer diagnosis having a high structural similarity index and resolution.

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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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Different modes of internalization of apoptotic alkyl-lysophospholipid and cell-rescuing lysophosphatidylcholine

Biochemical Journal ◽

10.1042/bj20030179 ◽

2003 ◽

Vol 374 (3) ◽

pp. 747-753 ◽

Cited By ~ 37

Author(s):

Arnold H. van der LUIT ◽

Marianne BUDDE ◽

Marcel VERHEIJ ◽

Wim J. van BLITTERSWIJK

Keyword(s):

Lipid Rafts ◽

De Novo ◽

Structural Similarity ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Chemotherapeutic Drugs ◽

Alternative Substrate ◽

High Structural Similarity ◽

Additional Cell ◽

Cell Membrane Level

The synthetic alkyl-lysophospholipid (ALP), Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), can induce apoptosis in tumour cells. Unlike conventional chemotherapeutic drugs, ALP acts at the cell-membrane level. We have reported previously that ALP is internalized, and interferes with phosphatidylcholine (PC) biosynthesis de novo, which appeared to be essential for survival in lymphoma cells [Van der Luit, Budde, Ruurs, Verheij and Van Blitterswijk (2002) J. Biol. Chem. 277, 39541–39547]. Here, we report that, in HeLa cells, ALP accumulates in lipid rafts, and that internalization is inhibited by low temperature, monensin, disruption of lipid rafts and expression of a dominant-negative mutant of dynamin bearing a replacement of Lys44 with alanine (K44A). Thus ALP is internalized via raft- and dynamin-mediated endocytosis. Dynamin-K44A alleviated the ALP-induced inhibition of PC synthesis and rescued the cells from apoptosis induction. Additional cell rescue was attained by exogenous lysoPC, which after internalization serves as an alternative substrate for PC synthesis (through acylation). Unlike ALP, and despite the high structural similarity to ALP, lysoPC uptake did not occur via lipid rafts and did not depend on functional dynamin, indicating no involvement of endocytosis. Albumin back-extraction experiments suggested that (radiolabelled) lysoPC undergoes transbilayer movement (flipping). We conclude that ALP is internalized by endocytosis via lipid rafts to cause apoptosis, while exogenous cell-rescuing lysoPC traverses the plasma membrane outside rafts by flipping. Additionally, our data imply the importance of ether bonds in lyso-phospholipids, such as in ALP, for partitioning in lipid rafts.

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