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1.
Yong-Hak Kim Woo-Seok Song Hayoung Go Chang-Jun Cha Cheolju Lee Myeong-Hee Yu Peter C. K. Lau Kangseok Lee 《Journal of bacteriology》2013,195(2):180-192
2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5′-dithio-bis-(2-nitrobenzoic acid) and ZnCl2, which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H2O2. SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions. 相似文献
2.
《Journal of molecular biology》2014,426(24):3985-4001
Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) subfamily of cytoplasmic tyrosine kinases. The C-terminal Pyk2-focal adhesion targeting (FAT) domain binds to paxillin, an adhesion molecule. Paxillin has five leucine-aspartate (LD) motifs (LD1–LD5). Here, we show that the second LD motif of paxillin, LD2, interacts with Pyk2-FAT, similar to the known Pyk2-FAT/LD4 interaction. Both LD motifs can target two ligand binding sites on Pyk2-FAT. Interestingly, they also share similar binding affinity for Pyk2-FAT with preferential association to one site relative to the other. Nevertheless, the LD2-LD4 region of paxillin (paxillin133 -290) binds to Pyk2-FAT as a 1:1 complex. However, our data suggest that the Pyk2-FAT and paxillin complex is dynamic and it appears to be a mixture of two distinct conformations of paxillin that almost equally compete for Pyk2-FAT binding. These studies provide insight into the underlying selectivity of paxillin for Pyk2 and FAK that may influence the differing behavior of these two closely related kinases in focal adhesion sites. 相似文献
3.
Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex
《Journal of molecular biology》2019,431(19):3647-3661
Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N1- and N3-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications. 相似文献
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Veremecheĭnko SN Vodianik MA Zdorovenko GM 《Prikladnaia biokhimiia i mikrobiologiia》2005,41(4):414-421
The structure and biological properties of lipopolysaccharides (LPSs) from strains IMB 4125 (=ATCC 13525) and IMB 7769 of the bacterium Pseudomonas fluorescens (biovar I) were studied in vitro. LPSs were similar in the composition of lipid A and the core lipid but differed in the structure of O-specific polysaccharide chains, which was corroborated by the absence of serological relationships between them. The toxicity (LD50) of LPSs of P. fluorescens with respect to D-glucosamine-sensitized mice was 40-50 times lower than the toxicity of the classic endotoxins, LPSs of E. coli. The LPSs studied stimulated the production of tumor necrosis factor (TNF) and nitric oxide (NO) by mouse peritoneal macrophages. The rates of TNF and NO synthesis induced by the LPSs of interest were eight to nine and three to five times lower, respectively, than the corresponding parameters of the control LPSs of E. coli 055:B5 and 026:B6. Additionally, LPS preparations of the P. fluorescens strains induced TNF synthesis by monocytes of human whole-blood preparations. Certain differences in biological properties of these strains have been revealed, which could be due to the characteristic features of LPS structure and composition in different cultures. 相似文献
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Yong-Xing He Liang Huang Yanyan Xue Xue Fei Yan-Bin Teng Sheryl B. Rubin-Pitel Huimin Zhao Cong-Zhao Zhou 《The Journal of biological chemistry》2010,285(7):4603-4611
2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C–C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 Å resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical α/β-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C–C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr121, Tyr229, and Asn132, which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well. 相似文献
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Structural Insights into the CFTR-NHERF Interaction 总被引:1,自引:0,他引:1
Ladias JA 《The Journal of membrane biology》2003,192(2):79-88
10.
Recently, it was identified that Pseudomonas aeruginosa competes with rival cells to gain a growth advantage using a novel mechanism that includes two interrelated processes as follows: employing type VI secretion system (T6SS) virulence effectors to lyse other bacteria, and at the same time producing specialized immunity proteins to inactivate their cognate effectors for self-protection against mutual toxicity. To explore the structural basis of these processes in the context of functional performance, the crystal structures of the T6SS virulence effector Tse1 and its complex with the corresponding immunity protein Tsi1 were determined, which, in association with mutagenesis and Biacore analyses, provided a molecular platform to resolve the relevant structural questions. The results indicated that Tse1 features a papain-like structure and conserved catalytic site with distinct substrate-binding sites to hydrolyze its murein peptide substrate. The immunity protein Tsi1 interacts with Tse1 via a unique interactive recognition mode to shield Tse1 from its physiological substrate. These findings reveal both the structural mechanisms for bacteriolysis and the self-protection against the T6SS effector Tse1. These mechanisms are significant not only by contributing to a novel understanding of niche competition among bacteria but also in providing a structural basis for antibacterial agent design and the development of new strategies to fight P. aeruginosa. 相似文献
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David A. Meekins Madushi Raththagala Kyle D. Auger Benjamin D. Turner Diana Santelia Oliver K?tting Matthew S. Gentry Craig W. Vander Kooi 《The Journal of biological chemistry》2015,290(38):23361-23370
Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates. 相似文献
16.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
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《Journal of molecular biology》2021,433(17):166995
Leak currents, defined as voltage and time independent flows of ions across cell membranes, are central to cellular electrical excitability control. The K2P (KCNK) potassium channel class comprises an ion channel family that produces potassium leak currents that oppose excitation and stabilize the resting membrane potential in cells in the brain, cardiovascular system, immune system, and sensory organs. Due to their widespread tissue distribution, K2Ps contribute to many physiological and pathophysiological processes including anesthesia, pain, arrythmias, ischemia, hypertension, migraine, intraocular pressure regulation, and lung injury responses. Structural studies of six homomeric K2Ps have established the basic architecture of this channel family, revealed key moving parts involved in K2P function, uncovered the importance of asymmetric pinching and dilation motions in the K2P selectivity filter (SF) C-type gate, and defined two K2P structural classes based on the absence or presence of an intracellular gate. Further, a series of structures characterizing K2P:modulator interactions have revealed a striking polysite pharmacology housed within a relatively modestly sized (~70 kDa) channel. Binding sites for small molecules or lipids that control channel function are found at every layer of the channel structure, starting from its extracellular side through the portion that interacts with the membrane bilayer inner leaflet. This framework provides the basis for understanding how gating cues sensed by different channel parts control function and how small molecules and lipids modulate K2P activity. Such knowledge should catalyze development of new K2P modulators to probe function and treat a wide range of disorders. 相似文献
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