1,721,014 research outputs found
STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF ESCHERICHIA COLI FLAVOHEMOGLOBIN.
Full text linkGIOVANNI ANTONINI, MAURIZIO PACI, NAZZARENO CAPITANI
Enzymatic, stereoselective synthesis of (S)-norcoclaurine.
No full textAn efficient, stereoselective, synthesis of (S)-norcoclaurine has been developed using the recombinant (S)-norcoclaurine synthase (NCS) enzyme, starting from tyrosine and dopamine substrates in a one-pot, two steps process1-3. The first step of the biotransformation consists in the oxidative decarboxylation of tyrosine (1) by stoichiometric amounts of sodium hypochlorite in order to generate 4-hydroxyphenylacetadehyde (2). In the second step NCS and dopamine (3) substrate are added in the reaction mix in the presence of ascorbate (necessary to prevent oxidation of the catechol moiety). Quantitative extraction of the newly formed (S)-norcoclaurine (4) from the aqueous solution was achieved by adsorption on active charcoal, dispersed in the reaction mixture. The optimized process afforded enantiomerically pure (S)-norcoclaurine (93 %) with a yield higher than 80% and allowed good recovery of the enzyme for recycling
The distal heme pocket of Escherichia coli flavohemoglobin probed by infrared spectroscopy
No full textThe infrared absorption spectra of ferric cyanide and ferrous carbonmonoxy Escherichia coli flavohemoglobin have been measured in order to probe the fine structural properties of the distal. heme pocket, characterized by the presence of a tyrosine in position B10 and a glutamine in position E7. The stretching frequency of iron bound cyanide occurs at 2136 cm(-1), an unusually high value if compared to other heme proteins. The infrared spectrum of the CO bound derivative displays two peaks centered at 1960 cm(-1) and at 1909 cm(-1) respectively. (H2O)-H-2 effects have been studied in both the ferric cyanide and ferrous CO derivatives in order to establish the presence of a distal hydrogen bonding to the iron bound ligand. The observed isotope shifts indicate that in the ferric cyanide derivative a hydrogen bond is donated from a residue in the distal pocket to the biatomic ligand whereas in the ferrous carbon monoxy derivative only the 1909 cm(-1) component is most likely hydrogen bonded to the phenolic group of TyrB10
Flavohemoglobin: structure and reactivity.
No full textFlavohemoglobins (flavoHbs) are made of a globin domain fused with a ferredoxin reductaselike FAD- and NAD-binding modules. These proteins are widely represented among bacteria and yeasts and represent a most challenging research subject in view of their high reactivity both as reductases and as oxidases. The functional annotations of flavoHbs are still controversial, and different physiological roles that are linked to cell responses to oxidative and/or nitrosative stress have been proposed. The flavoHb from Escherichia coli (HMP) has been the object of a large number of investigations to unveil its physiological role in the framework of bacterial resistance to nitrosative stress. HMP expression has been demonstrated to respond to the presence of NO in the culture medium, and an explicit mechanism has been proposed that involves NO scavenging and its reduction to N2O under anaerobic conditions. In contrast to (or together with) the anaerobic NO-reductase activity, HMP has also been shown to be able to catalyze the oxidation of NO to NO3- (NO-dioxygenase activity) both in vivo and in vitro in the presence Of 02 and NADH. HMP has also been shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. The alkylhydroperoxide reductase activity taken together with the unique lipid-binding properties of HMP suggests that this flavoHb may be involved in the repair of the lipid membrane oxidative damage generated during oxidative/ nitrosative stress
Electrocatalytic interconversion of NADH and NAD+ by Escherichia coli flavohemoglobin
No full textE. coli flavohemoglobin, oriented at electrodes via amphiphilic polymyxin B, electrocatalytically interconverts NADH and NAD(+) at its heme potentials operating as an electron transfer relay between the electrode and the protein FAD, where NADH/NAD(+) is transformed. The results are crucial for the development of NAD(+)-dependent bioelectrodes for biosynthesis, biosensors and biofuel cells.
PMID: 26389555 [PubMed - in process
A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca.
No full textThe gene coding for a hemoglobin-like protein (Tf-trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate-bound derivative, was obtained at 2.48 angstrom resolution. The three-dimensional structure of Tf-trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N- and C-terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far-UV CD melting experiments on the cyano-met derivatives. The ligand-binding properties of Tf-trHb, analyzed in stopped flow experiments, demonstrate that Tf-trHb is capable of efficient O-2 binding and release between 55 and 60 degrees C, the optimal growth temperature for Thermobifida fusca
Norcoclaurine Synthase: Mechanism of an Enantioselective Pictet-Spengler Catalyzing Enzyme
No full textThe use of bifunctional catalysts in organic synthesis finds inspiration in the selectivity of enzymatic catalysis which arises from the specific interactions between basic and acidic amino acid residues and the substrate itself in order to stabilize developing charges in the transition state. Many enzymes act as bifunctional catalysts using amino acid residues at the active site as Lewis acids and Lewis bases to modify the substrate as required for the given transformation. They bear a clear advantage over non-biological methods for their ability to tackle problems related to the synthesis of enantiopure compounds as chiral building blocks for drugs and agrochemicals. Moreover, enzymatic synthesis may offer the advantage of a clean and green synthetic process in the absence of organic solvents and metal catalysts. In this work the reaction mechanism of norcoclaurine synthase is described. This enzyme catalyzes the Pictet-Spengler condensation of dopamine with 4-hydroxyphenylacetaldehyde (4-HPAA) to yield the benzylisoquinoline alkaloids central precursor, (S)-norcoclaurine. Kinetic and crystallographic data suggest that the reaction mechanism occurs according to a typical bifunctional catalytic process
A gas chromatographic–mass spectrometric method for the simultaneous determination of resveratrol isomers and 2,4,6-trihydroxyphenanthrene in red wines exposed to UV-light
No full textResveratrol (3,5,4'-trihydroxystilbene) is one of the most abundant polyphenols in red grapes, and red wine represents one of the most important dietary source of this compound. Although its beneficial properties on human health have been widely investigated over the last 30 years, very little is known about its derivatives. Resveratrol can indeed undergo glycosylation, oligomerization and, upon UV-light exposure, it can isomerize from the trans- to the cis-isomer, which can further cyclize to 2,4,6-trihydroxyphenanthrene (THP). Although the effects of THP on human health are not yet known, being a polycyclic aromatic hydrocarbon, it can be potentially harmful. Since no data about THP occurrence in plant food and beverages are available, a simple procedure based on liquid-liquid extraction and GC-MS has been developed and validated for the simultaneous qualitative and quantitative analysis of trans-resveratrol, cis-resveratrol and THP in red wine, before and after UV-light exposure
Interaction with Membrane Lipids and Heme Ligand Binding Properties of Escherichia coli Flavohemoglobin.
No full textEscherichia coli flavohemoglobin has been shown to be able to bind specifically unsaturated and/or cyclopropanated fatty acids with very high affinity. Unsaturated or cyclopropanated fatty acid binding results in a modification of the visible absorption spectrum of the ferric heme, corresponding to a transition from a pentacoordinated (typical of the ligand free protein) to a hexacoordinated, high spin, heme iron. In contrast, no detectable interaction has been observed with saturated fatty acid, saturated phospholipids, linear, cyclic, and aromatic hydrocarbons pointing out that the protein recognizes specifically double bonds in cis conformation within the hydrocarbon chain of the fatty acid molecule. Accordingly, as demonstrated in gel filtration experiments, flavohemoglobin is able to bind liposomes obtained from lipid extracts of E. coli membranes and eventually abstract phospholipids containing cis double bonds and/or cyclopropane rings along the acyl chains. The presence of a protein bound lipid strongly affects the thermodynamic and kinetic properties of imidazole binding to the ferric protein and brings about significant modifications in the reactivity of the ferrous protein with oxygen and carbon monoxide. The effect of the bound lipid has been accounted for by a reaction scheme that involves the presence of two sites for the lipid/ligand recognition, namely, the heme iron and a non-heme site located in a loop region above the heme pocket
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