Biochemical, kinetic, and spectroscopic characterization of Ruegeria pomeroyi DddW - A mononuclear iron-dependent DMSP lyase

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW

Structural and Biochemical Insights into Dimethylsulfoniopropionate Cleavage by Cofactor-bound DddK from the Prolific Marine Bacterium Pelagibacter

Enormous amounts of the organic osmolyte dimethylsulfoniopropionate (DMSP) are produced in marine environments where bacterial DMSP lyases cleave it yielding acrylate and the climate-active gas dimethylsulfide (DMS). SAR11 bacteria are the most abundant clade of heterotrophic bacteria in the oceans and they play a key role in DMSP catabolism. An important environmental factor affecting DMS generation via DMSP lyases is the availability of metal ions since they are essential cofactors for many of these enzymes. Here we examine the structure and activity of DddK in the presence of various metal ions. We have established that DddK containing a double stranded β-helical motif utilizes various divalent metal ions as cofactors for catalytic activity. However, nickel, an abundant metal ion in marine environments, adopts a distorted octahedral coordination environment and conferred the highest DMSP lyase activity. Crystal structures of cofactor bound DddK reveal key metal ion binding and catalytic residues and provide the first rationalization for varying activities with different metal ions. The structures of DddK along with site-directed mutagenesis and UV-visible studies are consistent with Tyr 64 acting as a base to initiate the β-elimination reaction of DMSP. Our biochemical and structural studies provide a detailed understanding of DMS generation by one of the ocean’s most prolific bacterium

Spectroscopic and Computational Studies of Reduction of the Metal Versus the Tetrapyrrole Ring of Coenzyme F430 from Methyl-Coenzyme M Reductase

[[abstract]]Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this π-bond increases the overall π-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCRred1 state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120−13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77−89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states

Identification of residues involved in allosteric signal transmission from amino acid binding site of pyruvate kinase muscle isoform 2.

PKM2 is a rate-limiting enzyme in the glycolytic process and is involved in regulating tumor proliferation. Several amino acids (AAs) such as Asn, Asp, Val, and Cys have been shown to bind to the AA binding pocket of PKM2 and modulate its oligomeric state, substrate binding affinity, and activity. Although previous studies have attributed that the main chain and side chain of bound AAs are responsible for initiating signal to regulate PKM2, the signal transduction pathway remains elusive. To identify the residues involved in signal transfer process, N70 and N75 located at two ends of a β strand connecting the active site and AA binding pocket were altered. Biochemical studies of these variants with various AA ligands (Asn, Asp, Val, and Cys), illustrate that N70 and N75, along with β1 connecting these residues are part of the signal transduction pathway between the AA binding pocket and the active site. The results demonstrate that mutation of N70 to D prevents the transfer of the inhibitory signal mediated by Val and Cys, whereas N75 to L alteration blocks the activating signal initiated by Asn and Asp. Taken together, this study confirms that N70 is one of the residues responsible for transmitting the inhibitory signal and N75 is involved in the activation signal flow

Structural characterization and reactivity of Cu(II) complex of p-tert-butyl-calix[4]arene bearing two imine pendants at lower rim

Cu(II) complex of the double-armed p-tert-butylcalix[4]arene conjugate possessing imine functionality at the lower rim was synthesized and was characterized by analytical, spectral and crystallographic methods and its catecholase activity was evaluated.© Elsevie

Structural characterization and reactivity of Cu(II) complex of p-tert-butyl-calix[4]arene bearing two imine pendants at lower rim

Cu(II) complex of the double-armed p-tert-butylcalix[4]arene conjugate possessing imine functionality at the lower rim was synthesized and was characterized by analytical, spectral and crystallographic methods and its catecholase activity was evaluated

1,3-Di-peptido-conjugates of calix[4]arene and its di-OCH₃ derivatives: Synthesis, characterization and phosphate recognition

1098-1108Novel double-armed peptido-conjugates of calix[4]arene have been developed on the lower rim of the macrocycle. The functional group pendants exhibit conformational bend through the involvement of 11-atom N-H…O hydrogen bond inscribed in a 14-atom O-H…O interaction. As a result, only the terminal -COOR and -COOH groups are exposed to the environment, but not the amide moiety. The cone-conformation of the calix[4]arene is further stabilized through the O-H…O interactions at the lower rim. In effect, the conjugates exhibit a binding core at the lower rim along with hydrophobic cavity formed by the enclosure of arene moieties. Conformational mobility induced by the replacement of lower rim phenolic–OH by –OCH3 has also been demonstrated by variable temperature NMR studies in case of the corresponding –OCH3 derivatives. Differential receptor binding characteristics of these conjugates towards phosphate are demonstrated using absorption spectroscopy. The negatively charged phosphate group is received preferentially by the carboxylic terminal over the ester terminal conjugate

Mono-, di- and tri-nuclear Ni(II) complexes of N-, O-donor ligands: structural diversity and reactivity

A series of mono-, di- and tri-nuclear Ni(II) complexes of N, O-donating molecules possessing ---H2C---NH--- and ---HC=N--- moieties have been synthesized and characterized and the structures have been determined by single crystal X-ray diffraction. All these exhibited interesting molecular packing in their crystal lattices. Di-nuclear complexes were found to be cleaved in pyridine to result in mononuclear ones with additional coordinations being provided by pyridine. Di-nuclear complexes were found to form urea adducts as demonstrated based on absorption and vibrational studies.© Elsevie

Synthesis, structural diversity, inter-conversion and reactivity of Cu(II) complexes of hydroxy-rich molecules

Tetranuclear Cu(II) complexes having linear, cubane and pseudodouble-cubane cores were synthesized using hydroxy-rich molecules possessing amine and imine groups. The products were structurally characterized and were studied for their ability to oxidize catechol as well as for their inter-conversion between mono- and tetra-nuclear complexes

Structural comparisons of the binding cores formed by 1,3-di-Amide derivatives of p-tert-Butylcalix[4]arene: arms stabilization through Intra-molecular interactions including N-H***O, O-H***Cl and π***Cl types

his paper deals with the development of a receptor with selectively functionalized at the lower rim of p-tert-butyl calix[4]arene linked through amide bond, resulting in the diamide derivatives (L 1 , L 2 and L 3 ) suitable for sensing ions and molecules. All the derivatives were thoroughly characterized by analytical and spectral methods. These derivatives were structurally characterized by single crystal X-ray diffraction and the conformational features of the calix[4]arene as well as the pendants are discussed. The L 1 and L 2 exhibited intramolecular hydrogen bond interaction between the N-H and the lower rim phenolic oxygen in addition to the circular hydrogen bonding between the phenolic O-H and the ether oxygen of the adjacent strand. The corresponding H-bond is absent when no 'H' is present on amide-N as in case of L 3 . Such intramolecular N-H***O interactions observed with the pendant results in a conformational bend responsible for the orientation of the arms and there by the nature of the binding core formed. Thus the binding cores formed differ largely between L 3 and the other two