scholarly journals The tetrameric assembly of 2-aminomuconic acid dehydrogenase is a functional requirement of cofactor NAD+ binding

2021 ◽  
Author(s):  
Qiuli Shi ◽  
Yanjuan Chen ◽  
Xinxin Li ◽  
Hui Dong ◽  
Cheng Chen ◽  
...  
Keyword(s):  
Sole Source ◽  
Environmental Pollutant ◽  
Structural Basis ◽  
Metabolic Intermediate ◽  
Metabolizing Enzymes ◽  
Semialdehyde Dehydrogenase ◽  
X Ray Scattering ◽  
Quaternary Assembly ◽  
Single Operon ◽  
Tetrameric Assembly

The bacterium Pseudomonas sp. AP-3 is able to use the environmental pollutant 2-aminophenol as its sole source of carbon, nitrogen, and energy. Eight genes (amnA, B, C, D, E, F, G, and H) encoding 2-aminophenol metabolizing enzymes are clustered into a single operon. 2-aminomuconic 6-semialdehyde dehydrogenase (AmnC), a member of the aldehyde dehydrogenase (ALDH) superfamily, is responsible for oxidizing 2-aminomuconic 6-semialdehyde to 2-aminomuconate. In contrast to many other members of the ALDH superfamily, the structural basis of the catalytic activity of AmnC remains elusive. Here, we present the crystal structure of AmnC, which displays a homotetrameric quaternary assembly that is directly involved in its enzymatic activity. The tetrameric state of AmnC in solution was also presented using small-angle X-ray scattering. The tetramerization of AmnC is mediated by the assembly of a protruding hydrophobic beta-strand motif and residues V121 and S123 located in the NAD+-binding domain of each subunit. Dimeric mutants of AmnC dramatically lose NAD+ binding affinity and enzyme activity, indicating that tetrameric assembly of AmnC is required for oxidizing the unstable metabolic intermediate 2-aminomuconic 6-semialdehyde to 2-aminomuconic acid in the 2-aminophenol metabolism pathway.

scholarly journals Structural Basis of Cellulosome Efficiency Explored by Small Angle X-ray Scattering

2005 ◽  
Vol 280 (46) ◽  
pp. 38562-38568 ◽  
Author(s):  
Michal Hammel ◽  
Henri-Pierre Fierobe ◽  
Mirjam Czjzek ◽  
Vandana Kurkal ◽  
Jeremy C. Smith ◽  
...  
Keyword(s):  
Small Angle ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

scholarly journals Structural assembly of the human Miro1/2 GTPases based on the crystal structure of the N-terminal GTPase domain

2019 ◽  
Author(s):  
Kyle P. Smith ◽  
Pamela J. Focia ◽  
Srinivas Chakravarthy ◽  
Eric C. Landahl ◽  
Julian L. Klosowiak ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cancer Metastasis ◽  
Mitochondrial Dynamics ◽  
Structural Basis ◽  
Adapter Protein ◽  
Gtpase Domain ◽  
Saxs Data ◽  
Mitochondrial Regulation ◽  
X Ray Scattering ◽  
Structural Assembly

AbstractDysfunction in mitochondrial dynamics is believed to contribute to a host of neurological disorders and has recently been implicated in cancer metastasis. The outer mitochondrial membrane adapter protein Miro functions in the regulation of mitochondrial mobility and degradation, however, the structural basis for its roles in mitochondrial regulation remain unknown. Here, we report a 1.7Å crystal structure of N-terminal GTPase domain (nGTPase) of human Miro1 bound unexpectedly to GTP, thereby revealing a non-catalytic configuration of the putative GTPase active site. We identify two conserved surfaces of the nGTPase, the “SELFYY” and “ITIP” motifs, that are potentially positioned to mediate dimerization or interaction with binding partners. Additionally, we report small angle X-ray scattering (SAXS) data obtained from the intact soluble HsMiro1 and its paralog HsMiro2. Taken together, the data allow modeling of a crescent-shaped assembly of the full-length soluble domain of HsMiro1/2.PDB referenceCrystal structure of the human Miro1 N-terminal GTPase bound to GTP, 6D71

scholarly journals Homotaurine Metabolized to 3-Sulfopropanoate in Cupriavidus necator H16: Enzymes and Genes in a Patchwork Pathway

2009 ◽  
Vol 191 (19) ◽  
pp. 6052-6058 ◽  
Author(s):  
Jutta Mayer ◽  
Alasdair M. Cook
Keyword(s):  
Ralstonia Eutropha ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peptide Mass Fingerprinting ◽  
Sole Source ◽  
Cupriavidus Necator ◽  
Semialdehyde Dehydrogenase ◽  
Peptide Mass ◽  
Cupriavidus Necator H16 ◽  
Sulfoacetaldehyde Acetyltransferase

ABSTRACT Homotaurine (3-aminopropanesulfonate), a natural product and an analogue of GABA (4-aminobutyrate), was found to be a sole source of nitrogen for Cupriavidus necator (Ralstonia eutropha) H16, whose genome sequence is known. Homotaurine nitrogen was assimilated into cell material, and the quantitative fate of the organosulfonate was sulfopropanoate, which was recovered in the growth medium. The first scalar reaction was shown to be inducible homotaurine:2-oxoglutarate aminotransferase, which released 3-sulfopropanal from homotaurine. This aminotransferase was purified to homogeneity and characterized. Peptide mass fingerprinting yielded locus tag H16_B0981, which was annotated gabT, for GABA transaminase (EC 2.6.1.19). Inducible, NAD(P)+-coupled 3-sulfopropanal dehydrogenase, which yielded 3-sulfopropanoate from 3-sulfopropanal, was also purified and characterized. Peptide mass fingerprinting yielded locus tag H16_B0982, which was annotated gabD1, for succinate-semialdehyde dehydrogenase (EC 1.2.1.16). GabT and GabD1 were each induced during growth with GABA, and cotranscription of gabTD was observed. In other organisms, regulator GabC or GabR is encoded contiguous with gabTD: candidate GabR′ was found in strain H16 and in many other organisms. An orthologue of the GABA permease (GabP), established in Escherichia coli, is present at H16_B1890, and it was transcribed constitutively. We presume that GabR′PTD are responsible for the inducible metabolism of homotaurine to intracellular 3-sulfopropanoate. The nature of the exporter of this highly charged compound was unclear until we realized from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data that sulfoacetaldehyde acetyltransferase (EC 2.3.3.15; H16_B1872) was strongly induced during growth with homotaurine and inferred that the sulfite exporter encoded at the end of the gene cluster (H16_B1874) has a broad substrate range that includes 3-sulfopropanoate.

Structure of γ-conglutin: insight into the quaternary structure of 7S basic globulins from legumes

2015 ◽  
Vol 71 (2) ◽  
pp. 224-238 ◽  
Author(s):  
Jaroslaw Czubinski ◽  
Jakub Barciszewski ◽  
Miroslaw Gilski ◽  
Kamil Szpotkowski ◽  
Janusz Debski ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Quaternary Structure ◽  
Physiological Role ◽  
Structural Similarity ◽  
Disulfide Bridge ◽  
Structural Basis ◽  
Evolutionary Context ◽  
Lupin Seeds ◽  
High Level ◽  
Quaternary Assembly

γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteolytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated fromLupinus angustifoliusseeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and β subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographicD3symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a β-strand that is involved in intermolecular β-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.

scholarly journals An X-Ray Scattering Study into the Structural Basis of Corneal Refractive Function in an Avian Model

2013 ◽  
Vol 104 (12) ◽  
pp. 2586-2594 ◽  
Author(s):  
Siân R. Morgan ◽  
Erin P. Dooley ◽  
Paul M. Hocking ◽  
Chris F. Inglehearn ◽  
Manir Ali ◽  
...  
Keyword(s):  
Structural Basis ◽  
X Ray ◽  
X Ray Scattering ◽  
Scattering Study ◽  
Ray Scattering

scholarly journals The Structural Basis for BVMO Substrate Profile and Stereospecificity

2014 ◽  
Vol 70 (a1) ◽  
pp. C485-C485
Author(s):  
Brahm Yachnin ◽  
Michelle McEvoy ◽  
Roderick MacCuish ◽  
Krista Morley ◽  
Anthony Mittermaier ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Crystal Structures ◽  
Rational Design ◽  
Enzyme Mechanism ◽  
Structural Basis ◽  
Nmr Studies ◽  
X Ray Scattering ◽  
The Relationship ◽  
Villiger Oxidation

The Baeyer-Villiger monooxygenases (BVMOs) are a group of bacterial enzymes that are able to catalyze the synthetically useful Baeyer-Villiger oxidation reaction. As such, these enzymes have attracted considerable attention as potential industrial biocatalysts. The interest in these enzymes has led to a desire to be able to rationally design them for tailored biocatalytic applications. While recent years have seen the publication of a number of crystal structures (1-3), we have been lacking a structure of a BVMO that has its native substrate or product bound in a conformation that will allow the determination of substrate specificity and stereospecificity. Without such a structure, progress towards tailored BVMOs has been hampered. We have been able to solve two crystal structures of cyclohexanone monooxygenase (CHMO) with its lactone product, ε-caprolactone, bound. These structures place the lactone in an ideal position for the determination of its substrate specificity and stereospecificity. These structures have provided us with a better understanding of the structural basis for substrate binding, paving the way for the rational design of tailored BVMOs. At the same time, we have pursued small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) studies to better understand the dynamic nature of the enzyme. These studies have allowed us to explain the relationship between the various crystallized states of BVMOs and their complex, fourteen step enzyme mechanism.

scholarly journals Molecular packing structure of fibrin fibers resolved by X-ray scattering and molecular modeling

2020 ◽  
Author(s):  
Karin A. Jansen ◽  
Artem Zhmurov ◽  
Bart E. Vos ◽  
Giuseppe Portale ◽  
D. Hermida Merino ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Molecular Modeling ◽  
Molecular Packing ◽  
Structural Basis ◽  
X Ray ◽  
Packing Structure ◽  
X Ray Scattering ◽  
Wide Range ◽  
Blood Clots ◽  
Ray Scattering

ABSTRACTFibrin is the major extracellular component of blood clots and a proteinaceous hydrogel used as a versatile biomaterial. Fibrin forms branched networks of polymeric fibers, built of laterally associated double-stranded protofibrils. This multiscale hierarchical structure is crucial for the extraordinary mechanical resilience of blood clots. Yet, the structural basis of clot mechanical properties remains largely unclear due, in part, to the unresolved molecular packing structure of fibrin fibers. Here we quantitatively assess the packing structure of fibrin fibers by combining Small Angle X-ray Scattering (SAXS) measurements of fibrin networks reconstituted under a wide range of conditions with computational molecular modeling of fibrin oligomers. The number, positions, and intensities of the Bragg peaks observed in the SAXS experiments were reproduced computationally based on the all-atom molecular structure of reconstructed fibrin protofibrils. Specifically, the model correctly predicts the intensities of the reflections of the 22.5 nm axial repeat, corresponding to the half-staggered longitudinal arrangement of fibrin molecules. In addition, the SAXS measurements showed that protofibrils within fibrin fibers have a partially ordered lateral arrangement with a characteristic transverse repeat distance of 13 nm, irrespective of the fiber thickness. These findings provide fundamental insights into the molecular structure of fibrin clots that underlies their biological and physical properties.

scholarly journals Structural diversity of proline catabolic enzymes revealed by small angle x-ray scattering, x-ray crystallography and light scattering

2012 ◽  
Author(s):  
◽  
Ranjan Kumar Singh
Keyword(s):  
Small Angle ◽  
Quaternary Structure ◽  
Structural Basis ◽  
Sequence Alignments ◽  
X Ray ◽  
Catabolic Enzymes ◽  
Bifunctional Enzymes ◽  
X Ray Scattering ◽  
Proline Utilization ◽  
Ray Scattering

The proline catabolic enzymes catalyze the 4-electron oxidation of proline to glutamate. The reaction involves two enzymes, proline dehydrogenase (PRODH) and Î"1-pyrroline -5-carboxylate dehydrogenase (P5CDH). Some bacterial organisms have both of these enzymes fused together, and the fused bifunctional enzymes are called Proline utilization A (PutA). In addition to these bifunctional enzymes, some PutAs are trifunctional, because they moonlight as transcription repressors of their own gene. Our lab recently reported that the quaternary structure of the bifunctional PutA from B. japonicam (BjPutA) is a ring-shaped tetramer. However, the structural organization of PutAs from other organisms is still unknown. In particular, there are no structures available for moonlighting trifunctional PutAs. We therefore utilized small angle X-ray scattering (SAXS) to obtain the overall shape of a trifunctional PutA from Escherichia coli (EcPutA). In addition, rigid body modeling of full-length PutA has been done with the help of SAXS data and crystal structures of DNA-binding and PRODH domains of EcPutA, and BjPutA crystal structure. Unique structural features of PutA have also been explored through multiple sequence alignments and homology modeling using the webservers like ClustalW, Espript, Phyre, and Swiss Model. The results obtained from sequence alignment study led us to work on finding the diversity in oligomeric states of PutAs. Finally, the structural basis of HPII disease that is related to disorder in human P5CDH was determined through X-ray crystallographic studies.

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