排序方式: 共有92条查询结果,搜索用时 15 毫秒
31.
Milko Velarde Markus Hilberg Shang-Min Tu Antonio J. Pierik Wolfgang Buckel Albrecht Messerschmidt 《Journal of molecular biology》2009,391(3):609-553
3-Methylitaconate-Δ-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher Km but > 1000-fold lower kcat. The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 Å. The asymmetric unit of the P212121 unit cell with parameters a = 53.1 Å, b = 142.3 Å, and c = 228.4 Å contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an α/β-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Δ-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate. 相似文献
32.
Osipiuk J Mulligan R Bargassa M Hamilton JE Cunningham MA Joachimiak A 《The Journal of biological chemistry》2012,287(23):19452-19461
The crystal structure of SO1698 protein from Shewanella oneidensis was determined by a SAD method and refined to 1.57 Å. The structure is a β sandwich that unexpectedly consists of two polypeptides; the N-terminal fragment includes residues 1–116, and the C-terminal one includes residues 117–125. Electron density also displayed the Lys-98 side chain covalently linked to Asp-116. The putative active site residues involved in self-cleavage were identified; point mutants were produced and characterized structurally and in a biochemical assay. Numerical simulations utilizing molecular dynamics and hybrid quantum/classical calculations suggest a mechanism involving activation of a water molecule coordinated by a catalytic aspartic acid. 相似文献
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34.
NMR reveals structural rearrangements associated to substrate insertion in nucleotide‐adding enzymes 下载免费PDF全文
Biswaranjan Mohanty Michael Geralt Kurt Wüthrich Pedro Serrano 《Protein science : a publication of the Protein Society》2016,25(4):917-925
The protein NP_344798.1 from Streptococcus pneumoniae TIGR4 exhibits a head and base‐interacting neck domain architecture, as observed in class II nucleotide‐adding enzymes. Although it has less than 20% overall sequence identity with any member of this enzyme family, the residues involved in substrate‐recognition and catalysis are highly conserved in NP_344798.1. NMR studies showed binding affinity of NP_344798.1 for nucleotides and revealed μs to ms time scale rate processes involving residues constituting the active site. The results thus obtained indicate that large‐amplitude rearrangements of regular secondary structures facilitate the penetration of the substrate into the occluded nucleotide‐binding site of NP_344798.1 and, by inference based on sequence and structural homology, probably a wide range of other nucleotide‐adding enzymes. 相似文献
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36.
Shin DH Kim JS Yokota H Kim R Kim SH 《Protein science : a publication of the Protein Society》2006,15(4):921-928
We have determined the crystal structure of the DUF16 domain of unknown function encoded by the gene MPN010 of Mycoplasma pneumoniae at 1.8 A resolution. The crystal structure revealed that this domain is composed of two separated homotrimeric coiled-coils. The shorter one consists of 11 highly conserved residues. The sequence comprises noncanonical heptad repeats that induce a right-handed coiled-coil structure. The longer one is composed of approximately nine heptad repeats. In this coiled-coil structure, there are three distinguishable regions that confer unique structural properties compared with other known homotrimeric coiled-coils. The first part, containing one stutter, is an unusual phenylalanine-rich region that is not found in any other coiled-coil structures. The second part is a highly conserved glutamine-rich region, frequently found in other trimeric coiled-coil structures. The last part is composed of prototype heptad repeats. The phylogenetic analysis of the DUF16 family together with a secondary structure prediction shows that the DUF16 family can be classified into five subclasses according to N-terminal sequences. Based on the structural comparison with other coiled-coil structures, a probable molecular function of the DUF16 family is discussed. 相似文献
37.
Abhinav Kumar Marco Punta Herbert L Axelrod Debanu Das Carol L Farr Joanna C Grant Hsiu-Ju Chiu Mitchell D Miller Penelope C Coggill Heath E Klock Marc-André Elsliger Ashley M Deacon Adam Godzik Scott A Lesley Ian A Wilson 《Protein science : a publication of the Protein Society》2014,23(10):1380-1391
Crystal structures of three members (BACOVA_00364 from Bacteroides ovatus,
BACUNI_03039 from Bacteroides uniformis and BACEGG_00036 from Bacteroides
eggerthii) of the Pfam domain of unknown function (DUF4488) were determined to 1.95, 1.66,
and 1.81 Å resolutions, respectively. The protein structures adopt an eight-stranded,
calycin-like, β-barrel fold and bind an endogenous unknown ligand at one end of the
β-barrel. The amino acids interacting with the ligand are not conserved in any other protein
of known structure with this particular fold. The size and chemical environment of the bound ligand
suggest binding or transport of a small polar molecule(s) as a potential function for these
proteins. These are the first structural representatives of a newly defined PF14869 (DUF4488) Pfam
family. 相似文献
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The Shisa family of single-transmembrane proteins is characterized by an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Its founding member, Xenopus Shisa, promotes head development by antagonizing Wnt and FGF signaling. Recently, a mouse brain-specific Shisa protein CKAMP44 (Shisa9) was shown to play an important role in AMPA receptor desensitization. We used sequence similarity searches against protein, genome and EST databases to study the evolutionary origin and phylogenetic distribution of Shisa homologs. In addition to nine Shisa subfamilies in vertebrates, we detected distantly related Shisa homologs that possess an N-terminal domain with six conserved cysteines. These Shisa-like proteins include FAM159 and KIAA1644 mainly from vertebrates, and members from various bilaterian invertebrates and Porifera, suggesting their presence in the last common ancestor of Metazoa. Shisa-like genes have undergone large expansions in Branchiostoma floridae and Saccoglossus kowalevskii, and appear to have been lost in certain insects. Pattern-based searches against eukaryotic proteomes also uncovered several other families of predicted single-transmembrane proteins with a similar cysteine-rich domain. We refer to these proteins (Shisa/Shisa-like, WBP1/VOPP1, CX, DUF2650, TMEM92, and CYYR1) as STMC6 proteins (single-transmembrane proteins with conserved 6 cysteines). STMC6 genes are widespread in Metazoa, with the human genome containing 17 members. Frequent occurrences of PY motifs in STMC6 proteins suggest that most of them could interact with WW-domain-containing proteins, such as the NEDD4 family E3 ubiquitin ligases, and could play critical roles in protein degradation and sorting. STMC6 proteins are likely transmembrane adaptors that regulate membrane proteins such as cell surface receptors. 相似文献
40.
DUF647 (Domain of unknown function 647) 蛋白家族是在真核生物中广泛存在的、高度保守的蛋白家族。拟南芥中该基因家族共有6个成员, 迄今为止拟南芥DUF647家族中4个成员的功能尚不清楚。文章以拟南芥内源MIR319a前体为骨架, 构建了敲减DUF647家族中2个基因At1g13770和At2g23470表达的人工microRNAs(Artifical microRNAs, amiRNAs)。利用WMD(Web microRNA designer)平台设计分别靶向At1g13770和At2g23470基因的amiRNAs序列, 通过重叠PCR置换拟南芥MIR319a前体序列。构建融合amiRNAs前体的植物表达载体pCHF3-amiRNAs, 在农杆菌介导下转化拟南芥。RT-PCR分析表明, amiRNAs能够显著抑制At1g13770和At2g23470基因的表达, 获得了抑制效果明显的转基因株系。At2g23470-amiRNA转基因植株At2g23470转录水平的下调导致育性严重下降。文章为进一步研究这两个基因的功能奠定了良好的基础。 相似文献