Biochemical and Biophysical Characterization of the Human Ethylmalonic Encephalopathy non-Heme Sulfur [Fe]-Dioxygenase ETHE1, and X-ray Absorption Spectroscopy Applications and Methods Development

This work focuses on the biochemical and biophysical characterization of highly interesting metallo-proteins and is divided in two main projects. The first project regards the characterization of the iron-based enzyme ETHE1 from Homo sapiens, which is fundamental for detoxification from highly reactive persulfide species and whose specific mutations on the structural gene or on its regulatory sequences cause ethylmalonic encephalopathy. Moreover, ETHE1 was reported to be involved in apoptosis and cancer through the interaction with transcription factors such as NF-kB and p53. The protein has been studied by a number of techniques, noticeably XAS (Xray-absorption spectroscopy), SAXS (Small Angle X-ray Scattering Spectroscopy), Mössbauer spectroscopy and ITC (Iso-Thermal Calorimetry). The second project is based on XAS analysis and method development applied to metallo-proteins that are likely to have a substantial impact on basic and applied science. The mononuclear [Fe]-hydrogenase Hmd from Methanocaldococcus jannaschii has been extensively investigated by XANES and EXAFS analysis in the native and inhibited forms. This study revealed the major determiners of the electronic structure of this unique hydrogenase iron site and shed light on the chemicophysical requirements for the heterolysis of molecular hydrogen. In addition, I contribute to the characterization of the optically excited state of a bis (μ-oxo)-dicopper(III) complex mimicking the potential di-oxo to μ-oxo transition in tyrosinases active site. We took advantage of two cooperative approaches: pumped-XAS and an innovative combination of EXAFS spectroscopy and resonant Raman scattering. I present also the XAS analysis results obtained on the protein ABCE1, which is one of the most conserved proteins in the evolution of eukaryotes and archaea. ABCE1 is essential for cell viability and is a unique ATP-binding-cassette protein bearing iron-sulfur clusters

Hydrogen-activation mechanism of [Fe] hydrogenase revealed by multi-scale modeling

When investigating the mode of hydrogen activation by [Fe] hydrogenases, not only the chemical reactivity at the active site is of importance but also the large-scale conformational change between the so-called open and closed conformations, which leads to a special spatial arrangement of substrate and iron cofactor. To study H2 activation, a complete model of the solvated and cofactor-bound enzyme in complex with the substrate methenyl-H4MPT+ was constructed. Both the closed and open conformations were simulated with classical molecular dynamics on the 100 ns time scale. Quantum-mechanics/molecular-mechanics calculations on snapshots then revealed the features of the active site that enable the facile H2 cleavage. The hydroxyl group of the pyridinol ligand can easily be deprotonated. With the deprotonated hydroxyl group and the structural arrangement in the closed conformation, H2 coordinated to the Fe center is subject to an ionic and orbital push-pull effect and can be rapidly cleaved with a concerted hydride transfer to methenyl-H4MPT+. An intermediary hydride species is not formed

A Novel Secretory Poly-Cysteine and Histidine-Tailed Metalloprotein (Ts-PCHTP) from Trichinella spiralis (Nematoda)

BACKGROUND: Trichinella spiralis is an unusual parasitic intracellular nematode causing dedifferentiation of the host myofiber. Trichinella proteomic analyses have identified proteins that act at the interface between the parasite and the host and are probably important for the infection and pathogenesis. Many parasitic proteins, including a number of metalloproteins are unique for the nematodes and trichinellids and therefore present good targets for future therapeutic developments. Furthermore, detailed information on such proteins and their function in the nematode organism would provide better understanding of the parasite-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report the identification, biochemical characterization and localization of a novel poly-cysteine and histidine-tailed metalloprotein (Ts-PCHTP). The native Ts-PCHTP was purified from T. spiralis muscle larvae that were isolated from infected rats as a model system. The sequence analysis showed no homology with other proteins. Two unique poly-cysteine domains were found in the amino acid sequence of Ts-PCHTP. This protein is also the first reported natural histidine tailed protein. It was suggested that Ts-PCHTP has metal binding properties. Total Reflection X-ray Fluorescence (TXRF) assay revealed that it binds significant concentrations of iron, nickel and zinc at protein:metal ratio of about 1:2. Immunohistochemical analysis showed that the Ts-PCHTP is localized in the cuticle and in all tissues of the larvae, but that it is not excreted outside the parasite. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Ts-PCHTP is the first described member of a novel nematode poly-cysteine protein family and its function could be metal storage and/or transport. Since this protein family is unique for parasites from Superfamily Trichinelloidea its potential applications in diagnostics and treatment could be exploited in future

Characterization and 1.57 \uc5 resolution structure of the key fire blight phosphatase AmsI from Erwinia amylovora

AmsI is a low-molecular-weight protein tyrosine phosphatase that regulates the production of amylovoran in the Gram-negative bacterium Erwinia amylovora, a specific pathogen of rosaceous plants such as apple, pear and quince. Amylovoran is an exopolysaccharide that is necessary for successful infection. In order to shed light on AmsI, its structure was solved at 1.57 \uc5 resolution at the same pH as its highest measured activity (pH 5.5). In the active site, a water molecule, bridging between the catalytic Arg15 and the reaction-product analogue sulfate, might be representative of the water molecule attacking the phospho-cysteine intermediate in the second step of the reaction mechanism.The high-resolution (1.57 \uc5) structure and kinetic data of the key fire blight phosphatase AmsI are reported

A model-based proposal for the role of UreF as a GTPase activating protein in the urease active site biosynthesis

UreF is a protein that plays a role in the in vivo urease activation as a chaperone involved in the insertion of two Ni2+ ions in the apo-urease active site. The molecular details of this process are unknown. In the absence of any molecular information on the UreF protein class, and as a step toward the comprehension of the relationships between UreF function and structure, we applied a structural modeling approach to infer useful biochemical knowledge on Bacillus pasteurii UreF (BpUreF). Similarity searches and multiple alignment of UreF protein sequences indicated that this class of proteins has a low homology with proteins of known structure. Fold recognition methods were therefore used to identify useful protein structural templates to model the structure of BpUreF. In particular, the templates belong to the class of GTPase-activating proteins. Modeling of BpUreF based on these templates was performed using the program MODELLER. The structure validation yielded good statistics, indicating that the model is plausible. This result suggests a role for UreF in urease active site biosynthesis as a regulator of the activity of UreG, a small G protein involved in the in vivo apo-urease activation process and established to catalyze GTP hydrolysis

Crystal Structures of [Fe]-Hydrogenase in Complex with Inhibitory Isocyanides: Implications for the H₂-Activation Site

Inhibition mechanism: Isocyanides strongly inhibit [Fe]-hydrogenase. X-ray crystallography and X-ray absorption spectroscopy revealed that the isocyanide binds to the trans position, versus the acyl carbon of the Fe center, and is covalently bound to the pyridinol hydroxy oxygen. These results also indicated that the hydroxy group is essential for H2 activation

Comparison of the Levansucrase from the epiphyte Erwinia tasmaniensis vs its homologue from the phytopathogen Erwinia amylovora

Erwinia tasmaniensis is an epiphytic bacterium related to the plant pathogen Erwinia amylovora, the etiological agent of fire blight. In this study the levansucrase from E. tasmaniensis (EtLsc) has been compared with the homologous enzyme from E. amylovora (EaLsc). We characterized the enzymatic activity and compared the products profile of both enzymes by High Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detector (HPAEC-PAD). Moreover we determined the crystal structure of EtLsc to understand the structural peculiarity causing the different product profiles of the two homologues. EtLsc exhibits increased efficiency in the production of FOS, resulting in a better catalyst for biotechnological synthesis than EaLsc. Based on our results, we propose that the role of this enzyme in the life cycle of the two bacteria is most likely related to survival, rather than linked to pathogenicity in E. amylovora

Structural and functional analysis of Erwinia amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of D-sorbitol 6-phosphate to D-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Å resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing D-sorbitol 6-phosphate than in reducing D-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the D-sorbitol 6-phosphate present in the in vitro environment is converted into D-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding