Denis Thibaut

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4‐dimethylamino‐l‐phenylalanine precursor of pristinamycin I

Four pap genes (papA, papB, papC, papM ) were found by sequencing near to snbA, a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4‐dimethylamino‐l‐phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI− phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N‐methylation steps of 4‐amino‐l‐phenylalanine leading to DMPAPA via 4‐methylamino‐l‐phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.

Biosynthesis of vitamin B12: the structure of factor IV, the oxidized form of precorrin-4

13 C Labelling and NMR experiments establish the structure of factor IV, the oxidized form of the tetramethylated intermediate on the biosynthetic pathway to precorrin-6x 2 in Pseudomonas denitrificans, thus demonstrating that the contraction of the macrocycle leads to an acetyl derivative at C-1.

Biosynthesis of vitamin B12: structure of precorrin-3B, the trimethylated substrate of the enzyme catalysing ring contraction

FAB-MS, FTIR spectroscopy, 13C labelling and NMR experiments establish the structure of precorrin-3B, a further trimethylated intermediate along the vitamin B12 pathway, thus demonstrating that precorrin-3B is synthesised from precorrin-3 (called precorrin-3A from now) by a complex oxidative reaction involving C-20 hydroxylation and γ-lactone formation from ring-A acetate to C-1, catalysed by the CobG protein in Pseudomonas denitrificans.

Biosynthesis of vitamin B12: structural studies on precorrin-8x, an octamethylated intermediate and the structure of its stable tautomer

The structure of precorrin-8x, the octamethylated B12-intermediate which follows precorrin-6y in Pseudomonas denitrificans, is largely determined by 13C-labelling and NMR spectroscopy and the structure of its most stable tautomer is established.

Biosynthesis of vitamin B12 : the site of reduction of precorrin-6x

It is proved by deuterium labelling and NMR spectroscopy that a hydride equivalent from NADPH is transferred to C-19 of precorrin-6x as this intermediate is converted enzymically into hydrogenobyrinic acid.

Biosynthesis of vitamin B12: stereochemistry of transfer of a hydride equivalent from NADPH by precorrin-6x reductase

Deuterium labelling with NMR analysis demonstrates that the enzyme precorrin-6x reductase transfers HR from C-4 of the pyridine residue of NADPH as the first step in the sequence leading from precorrin-6x to hydrogenobyrinic acid.

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.

Biosynthesis of vitamin B12: incorporation of (11S)-[11-2H1-] and (11R)-[11-2H1]porphobilinogen into sirohydrochlorin and 2,7,20-trimethylisobacteriochlorin

(11S)-[11-2H1]Porphobilinogen (1a) and the (11R)-isomer (1b) are incorporated biosynthetically into precorrin-2 and precorrin-3 which are isolated as the aromatised esters (7) and (9) for 1H NMR studies; the unexpected results are discussed.