Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135350/1/feb212479.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/135350/2/feb212479_am.pd

Structural comparison of the cytochrome P450 enzymes CYP106A1 and CYP106A2 provides insight into their differences in steroid conversion

Understanding the structural basis of the selectivity of steroid hydroxylation requires detailed structural and functional investigations on various steroid hydroxylases with different selectivities, such as the bacterial cytochrome P450 enzymes. Here, the crystal structure of the cytochrome P450 CYP106A1 from Priestia megaterium was solved. CYP106A1 exhibits a rare additional structural motif of a cytochrome P450, a sixth β-sheet. The protein was found in different unusual conformations corresponding to both open and closed forms even when crystallized without any known substrate. The structural comparison of CYP106A1 with the previously investigated CYP106A2, including docking studies for both isoforms with the substrate cortisol, reveals a completely different orientation of the steroid molecule in the active sites. This distinction convincingly explains the experimentally observed differences in substrate conversion and product formation by the two enzymes

Structural characterization of the thermostable <i>Bradyrhizobium japonicum</i> D-sorbitol dehydrogenase

Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is d-glucitol (a synonym for d-sorbitol), although l-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with d-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T (m) for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119- dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119- dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior–Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse

Structural and functional analysis of galactitol-dehydrogenase from Rhodobacter sphaeroides and the thiol disulfide oxidoreductase SoxS from Paracoccus pantotrophus

Ziel der Arbeit war die Struktur-Funktionsanalyse zweier bakterieller Redox-Enzyme. Die Galaktitol-Dehydrogenase (GatDH) aus Rhodobacter sphaeroides ist in der Lage, enantioselektiv sowohl ein breites Spektrum an Polyolen zu oxidieren als auch umgekehrt Ketone zu reduzieren. Die Struktur von GatDH konnte, in Komplex mit dem Kofaktor, geklärt werden. Um bessere Einsicht in die Substratbindung zu bekommen, wurden weitere Strukturen mit im aktiven Zentrum gebundenen Substraten gelöst. Der vorgeschlagene Reaktionsweg der GatDH entspricht der für die meisten SDR-Enzyme postulierten Katalyse. GatDH bildet im Kristall und in Lösung stabile Tetramere, was durch Lichtstreuungs-Experimente verifiziert werden konnte. Die periplasmatische Thiol-Disulfid-Oxidoreduktase SoxS ist für den Schwefel-oxidierenden (Sox) Phänotyp des chemotrophen Bakteriums Paracoccus pantotrophus wichtig. Die Struktur von SoxS konnte in der oxidierten als auch in der reduzierten Form aufgeklärt werden. SoxS zeigt eine hohe strukturelle Homologie zu typischen cytoplasmatischen bakteriellen Thioredoxinen. Dagegen besitzt SoxS im aktiven Zentrum ein Sequenzmotiv, welches nicht in anderen Thioredoxinen vorkommt, aber eng verwandt mit den Sequenzen einiger Glutaredoxinen und DsbC-/DsbG-Mitgliedern ist. SoxS kombiniert in Struktur, Substratspezifität und Reaktionsweise Eigenschaften von Thioredoxinen, Glutaredoxinen und von Mitgliedern der Thiol-Disulfid-Oxidoreduktasen der Dsb-Familie.Aim of this work was the structural and functional characterization of two bacterial redox-enzymes. The galactitol-dehydrogenase (GatDH) of the purple bacterium Rhodobacter sphaeroides is able to oxidize a wide spectrum of polyols as well as the reduction of ketones. The structure of GatDH in presence of its cofactor could be determined. To gain deeper insight in the specificity of substrate binding further structures with bound substrates were solved. The proposed reaction pathway of GatDH corresponds to the catalysis postulated for most SDR enzymes. GatDH forms in the crystal as well as in solution stable tetramers as verified by light scattering experiments. The periplasmic thiol-disulfide oxidoreductase SoxS is beneficial for the sulphur oxidizing (Sox) phenotype of the chemotrophic bacterium Paracoccus pantotrophus. The structure of SoxS was solved in its oxidized and reduced form, respectively. SoxS reveals a high structural homology to typical cytoplasmic bacterial thioredoxins. By contrast, SoxS contains the active site motif Pro-Gly-Cys-Leu-Tyr-Cys, not present in other thioredoxins. Interestingly, the sequence of this motif is closely related to the sequence of some glutaredoxins and to the sequences of some members of the thiol-disulfide oxidoreductases DsbC and DsbG. SoxS combines features of thioredoxins, glutaredoxins and the thiol-disulfide oxidoreductases of the Dsb-family in structure, target specificity and reaction

Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides.

The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg(2+) and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix alpha(1) in full-length ABCB6

Structural features of chloroplast trigger factor determined at 2.6 Å resolution

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis–trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Å resolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts

Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell