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J L Ferrer

Publications and source records attributed to J L Ferrer.

8 recordsLinked to original sources
PubMed2000

Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module.

Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.

Amino Acid Sequence↗
PubMed2000

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase.

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.

Acetyl Coenzyme A↗
PubMed1999

Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis.

Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants. It produces chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters into a polyketide reaction intermediate that cyclizes. The crystal structures of CHS alone and complexed with substrate and product analogs reveal the active site architecture that defines the sequence and chemistry of multiple decarboxylation and condensation reactions and provides a molecular understanding of the cyclization reaction leading to chalcone synthesis. The structure of CHS complexed with resveratrol also suggests how stilbene synthase, a related enzyme, uses the same substrates and an alternate cyclization pathway to form resveratrol. By using the three-dimensional structure and the large database of CHS-like sequences, we can identify proteins likely to possess novel substrate and product specificity. The structure elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products.

Acyltransferases↗
PubMed1998

Data compression for diffraction patterns.

Efficient coding (lossless) and compression (lossy) of diffraction patterns is important in protein crystallography experiments because of storage and transmission limitations. The goal is to reduce the bit-rate significantly while keeping diffraction peak intensity distortion at an acceptable level. This paper presents an overview of coding and compression techniques more or less adapted to such problems. A large part of this study is dedicated to time-frequency-transform based compression algorithms and some of their extensions. Wavelet based software has been developed and tested. Results are compared with the discrete cosine transform (DCT) and other classical algorithms. These tools seem attractive and very promising for analyzing and compressing signals with singularities and transient phenomena such as diffraction peaks. Tests were performed on a standard protein crystallography data set coming from the CCD detector of D2AM beamline at the European Synchrotron Radiation Facility at Grenoble. These were compressed with DCT and wavelet-based algorithms. It appears that alterations of the result of the processing of restored images remain very weak for compression rates up to 10. These preliminary results indicate that the proposed wavelet method is a good standard technique for efficient compression of diffraction patterns.

Algorithms↗
PubMedDate not supplied

Observations of middle ear pressures. Commentary with movie.

Previous direct measurements of middle ear pressure in ears with serous otitis resulted in the range of from 0 to -10 mm H2O pressure. To confirm these findings we attempted to quantify middle ear pressures by doing myringotomies in serous otitis patients through sterile saline solution. We compared the rate of aspiration of the saline in ears with serous otitis to the rate of aspiration of saline after an experimental myringotomy in an ear model in which known pressures were imposed. To record our findings we used motion picture photography. Layering a film of sterile oil suspension of oxytetracycline and hydrocortisone over dry tympanic membrane perforations resulted in the demonstration of a pulsatile positive pressure of about 6 mm water in many of the ears which we tested. The oil film often formed an external bubble which ruptured after several minutes. In some ears there was no change in pressure and in only a small percentage there was evidence of a decreased pressure by absorption of air in the middle ear during the period of observation. This positive pressure is unrelated to swallowing and suggests that the current theories of middle ear aeration via opening of the eustachian tube may not be valid. These findings were demonstrated with motion picture film.

Acoustic Impedance Tests↗