The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1 Å resolution. NCS shares no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-Å -long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.

The x-ray structure of ferric unliganded lipid-free Escherichia coli flavohemoglobin has been solved to a resolution of 2.2 Å and refined to an R-factor of 19%. The overall fold is similar to that of ferrous lipid-bound Alcaligenes eutrophus flavohemoglobin with the notable exception of the E helix positioning within the globin domain and a rotation of the NAD binding module with respect to the FAD-binding domain accompanied by a substantial rearrangement of the C-terminal region. An inspection of the heme environment in E. coli flavohemoglobin reveals an unexpected architecture of the distal pocket. In fact, the distal site is occupied by the isopropyl side chain Leu-E11 that shields the heme iron from the residues in the topological positions predicted to interact with heme iron-bound ligands, namely Tyr-B10 and Gln-E7, and stabilizes a pentacoordinate ferric iron species. Ligand binding properties are consistent with the presence of a pentacoordinate species in solution as indicated by a very fast second order combination rates with imidazole and azide. Surprisingly, imidazole, cyanide, and azide binding profiles at equilibrium are not accounted for by a single site titration curve but are biphasic and strongly suggest the presence of two distinct conformers within the liganded species.Flavohemoglobins are oxygen-binding proteins composed of a typical globin domain containing a B-type heme fused with a ferredoxin reductase-like FAD-and NAD-binding domain. Together with single domain bacterial hemoglobins and six-helices containing "truncated hemoglobins," flavohemoglobins are part of a vast family of "hemoglobin-like" proteins whose functions are still elusive. Because of the identification of flavohemoglobin genes in a wide variety of prokaryotic and eukaryotic microorganisms, a crescendo of experimental observations has revealed that these proteins possess unique structural and functional properties unrelated to those of hemoproteins involved in oxygen transport and storage (1).Escherichia coli flavohemoglobin (HMP) 1 is certainly the most extensively studied protein within the bacterial hemoglobin family. At present, the most credited physiological function of HMP has been inferred from the observation that protein expression in the bacterial cell is enhanced in the presence of nitric oxide releasers in the culture medium (2-4). Accordingly, HMP has been shown to be able to catalyze the oxidation of free nitric oxide to nitrate both in vivo and in vitro in the presence of oxygen and NADH (5). In this framework, HMP is thought to be involved in the response of the bacterial cell to the potentially harmful role of free nitric oxide in giving rise to nitrosative stress. Nevertheless, although the nitric-oxide dioxygenase activity stands as the major functional hypothesis for HMP, the molecular mechanism and the structural determinants at the basis of this enzymic function remain poorly understood. In particular, it has not been established as yet how the amino acid side chains in the distal heme pocket affect...

SummaryFlavohemoglobins (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 N 2 O 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 NO 3 2 (NO-dioxygenase activity) both in vivo and in vitro in the presence of O 2 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. NO-dioxygenase activity; repair of the lipid membrane oxidative damage; alkylhydroperoxide reductase activity.

The 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 Å 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 O2 binding and release between 55 and 60 °C, the optimal growth temperature for Thermobifida fusca.

An efficient, stereoselective, green synthesis of (S)-norcoclaurine (higenamine) has been developed using the recombinant (S)-norcoclaurine synthase (NCS) enzyme, starting from the cheap tyrosine and dopamine substrates in a one-pot, two step process. Key steps in the biotransformation consist of the oxidative decarboxylation of tyrosine by stoichiometric amounts of sodium hypochlorite in order to generate 4-hydroxyphenylacetadehyde, followed by the addition of enzyme and dopamine substrate in the presence of ascorbate, a necessary ingredient in order to avoid oxidation of the catechol moiety. Quantitative extraction of the product from an aqueous solution was achieved by adsorption onto active charcoal dispersed in the reaction mixture. The optimized process afforded enantiomerically pure (S)-norcoclaurine (93%) in a yield higher than 80% and allowed good recovery of the enzyme for recycling. The process thus developed represents the first example of a green Pictet-Spengler synthesis, which may pave the way to novel strategies in benzylisoquinoline alkaloid synthesis

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high affinity for hydrogen sulfide (K = 5.0 x 10(6) and 2.8 x 10(6) M(-1) for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though lower with respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 x 10(8) M(-1)). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH(-)-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH(-) whereas the YCD1 and YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm(-1) is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen sulfide is thought to have a possible physiological significance as H(2)S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.

Internal water molecules play an active role in ligand uptake regulation, since displacement of retained water molecules from protein surfaces or cavities by incoming ligands can promote favorable or disfavorable effects over the global binding process. Detection of these water molecules by X-ray crystallography is difficult given their positional disorder and low occupancy. In this work, we employ a combination of molecular dynamics simulations and ligand rebinding over a broad time range to shed light into the role of water molecules in ligand migration and binding. Computational studies on the unliganded structure of the thermostable truncated hemoglobin from Thermobifida fusca (Tf-trHbO) show that a water molecule is in the vicinity of the iron heme, stabilized by WG8 with the assistance of YCD1, exerting a steric hindrance for binding of an exogenous ligand. Mutation of WG8 to F results in a significantly lower stabilization of this water molecule and in subtle dynamical structural changes that favor ligand binding, as observed experimentally. Water is absent from the fully hydrophobic distal cavity of the triple mutant YB10F-YCD1F-WG8F (3F), due to the lack of residues capable of stabilizing it nearby the heme. In agreement with these effects on the barriers for ligand rebinding, over 97% of the photodissociated ligands are rebound within a few nanoseconds in the 3F mutant case. Our results demonstrate the specific involvement of water molecules in shaping the energetic barriers for ligand migration and binding.

Escherichia 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.