Didier Faucher

Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure.

An aliphatic nitrilase, active on adiponitrile and cyanovaleric acid, was identified and purified from Comamonas testosteroni sp. (Ct). Oligodeoxyribonucleotide probes were designed from limited amino acid (aa) sequence information and used to clone the corresponding gene, named nitA. High homologies were found at the aa level between Ct nitrilase and the sequences of known nitrilases. Multi-alignment of sequenced nitrilases suggests that Cys163 of Ct plays an essential role in the active site. This hypothesis is strengthened by molecular studies on nitrilases from Alcaligenes faecalis JM3, and Rhodococcus rhodochrous J1 and K22 [Kobayashi et al., Proc. Natl. Acad. Sci. USA 90 (1993) 247-251; J. Biol. Chem. 267 (1992) 20746-20751; Biochemistry 31 (1992) 9000-9007]. Large amounts of an active recombinant enzyme could be produced in Escherichia coli when nitA was overexpressed together with the E. coli groESL genes.

The major biotinyl protein from Pisum sativum seeds covalently binds biotin at a novel site

Seeds of Pisum sativum contain a biotinyl polypeptide called SBP65 that behaves as a putative sink for the free vitamin, representing more than 90% of the total protein-bound biotin in mature seeds. A cDNA encoding SBP65 was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing. Peptide sequencing also indicated binding of the biotin to lysine 103. The biotinylation domain of SBP65 differs markedly from that of presently known biotin enzymes. Molecular analysis of the protein sequence reveals an extremely hydrophilic protein containing several repeated motifs. These properties, as well as the temporal and spatial patterns of expression of this protein, suggest that SBP65 belongs to the LEA (late embryogenesis-abundant) group of proteins.

Two rat homologues of human cystatin C

Two immunochemically related forms of cystatin C‐like inhibitors which differ in their M r app and isoelectric point have been found both in urine and seminal vesicles of rats. Amino‐terminal sequences of these two cystatins are identical within the same fluid and exhibit a high degree of homology with that of human cystatin C. However, cystatins C purified from urine lack eight residues at their amino‐terminal end when compared to those of seminal vesicles. The occurrence of two cystatin C‐like components in rat fluids has been found to be due to the presence of a glycosylated form reported here as cystatin Cg which specifically binds concanavalin A and is susceptible to endo‐β‐N‐acetylglucosaminidase treatment.

Low Molecular Weight, Sequence Based, Collagenase Inhibitors Selectively Block the Interaction Between Collagenase and TIMP (Tissue Inhibitor of Metalloproteinases)

Sequence-based inhibitors of collagenase bearing an hydroxamate group capable of chelating the active site zinc atom were synthesized and tested. The effect of one of these molecules (RP 59794; Ki about 10(-8) M) on the formation of the TIMP: collagenase complex was also tested. RP 59794 blocks complex formation and can partially dissociate established TIMP: collagenase complexes. It exhibits the same stereospecificity in this activity as in its inhibition of collagenase suggesting that TIMP and RP 59794 both interact with the active site region of collagenase.

Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing.

The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.

IDENTIFICATION OF THE CYTOSKELETAL PROTEIN ALPHA -ACTININ AS A PLATELET THROMBOSPONDIN-BINDING PROTEIN

Binding of the α‐granular thrombospondin (TSP) to the plasma membrane of activated platelets has long been documented, yet the molecular mechanism involved in its secretion and surface expression have not been elucidated. Using a ligand blot binding assay where electrophoretically separated platelet proteins were incubated with purified125I‐labeled TSP, we observed a strong interaction of [125I]TSP with a 100 kDa single chain protein. On performing a platelet subfractionation, the 100 kDa protein was predominantly localized in the cytosol from which it was purified by preparative electrophoresis and was identified by amino acid sequencing to the cytoskeletal protein, α‐actinin. We further demonstrated that [125I]TSP interacts with α‐actinin in a specific manner and with a high affinity (K d = 6.6nM) in a solid‐phase binding assay.

Enzymatic hydrolysis of adiponitrile into 5-cyano valeric acid, an intermediate for nylon 6

Publisher Summary An industrial process must be economic and safe for the environment. The chemical hydrolysis of nitrile in acid is well known. Nearly all nitriles react with either basic or acid catalysts, but considerable quantities of inorganic salts are always produced as by-products. The only way to suppress these by-products is to produce the ammonium carboxylate under neutral pH and then to recover the ammonia by dissociation of the salt between the weak base and acid. It is possible to design an excellent catalyst for a bulk product such as caprolactam. The first advantage of the biocatalysis in this route is able to carry out this reaction at neutral pH. The second is to selectively obtain the α,ω-cyanoacid starting from the α,ω-dinitrile. No other type of catalysis can do this.