Michel Gaudry

Biotin uptake: influx, efflux and countertransport in Escherichia coli K12.

Biotin uptake by Escherichia coli K12 has been reinvestigated. The vitamin uptake is an active process depending on energy and inhibited by uncouplers. The kinetic parameters (Km = 0.27 microM, Vmax = 6.8 pmol/min per mg dry cells) are close to those previously determined for a biotin-dependent strain E. coli C162 (Piffeteau, A., Zamboni, M. and Gaudry, M. (1982) Biochim. Biophys. Acta 688, 29-36). By use of biotin p-nitrophenyl ester, an affinity label of the biotin transport system, it was shown, under conditions of steady state, that the efflux of biotin is not energy dependent and is mainly mediated by a diffusion mechanism. Reexamination of the regulation of the biotin transport by biotin, revealed that only 50% of the biotin uptake system is under control by the vitamin.

Vitamin K-dependent carboxylation of synthetic substrates. Nature of the products.

Abstract Vitamin K-dependent carboxylation of synthetic Phe-Leu-Glu-Glu-Val by solubilized rat liver microsomes yields a predominant product Phe-Leu-Gla-Glu-Val, as determined by decarboxylation and acid or enzymatic hydrolysis. A small amount of another monocarboxylated product, yet unidentified, is also detected. This product is formed from Phe-Leu-Gla-Glu-Val in an enzymatic vitamin K-independent reaction.

Vitamin K dependent carboxylation: Study of diastereoisomeric γ-methylglutamic acid containing peptidic substrates

Two pentapeptides Phe-Leu-X-Glu-Val where X is either the L-threo-gamma-methylglutamic acid or the L-erythro isomer have been synthesized and tested as substrates in the vitamin K dependent carboxylation. The gamma-methylglutamic residue is not carboxylated and both peptides are inhibitors of the carboxylation of the reference peptide Phe-Leu-Glu-Glu-Val. The threo containing isomer has a much better affinity than the reference and is the best inhibitor of this reaction described so far.

Biotin transport by a biotin-deficient strain of escherichia coli

Biotin uptake has been investigated using an Escherichia coli biotin requiring auxotroph grown under biotin-deficient conditions. This strain accumulated biotin in the free and bound form. In agreement with a previous report by O. Prakash and M.A. Eisenberg (J. Bacteriol. 120 (1974) 785-791), the biotin entry proved to be an active process which depended on an energy source and was inhibited in the presence of uncouplers. The kinetic parameters have been determined (KM = 0.05 microM, Vmax = 7 pmol/min per mg dry weight). The pool of free biotin could be readily exchanged with external biotin and decreased to a very low level in the absence of an energy source. The use of several biotin analogues revealed that this transport system was quite specific for biotin: slight modifications, for instance in the valeric chain, lowered drastically the affinity for the carrier.

CHEMO-ENZYMIC SYNTHESIS OF PROTECTED CYANO DERIVATIVES OF GLUTAMATE

Abstract (2S,4RS)-Boc-4-cyanoglutamate γ-methyl ester (ee = 95%) and (2S)-Boc-2-amino-4-bis(cyano)butyrate (ee = 96%) were synthesised in respectively 43% and 40% overall yields by addition of sodium malonate derivatives onto Boc-dehydroalanine methyl ester followed by regio- and stereoselective hydrolysis of α-methyl ester by α-chymotrypsin. These regio-and stereoselectivities were strongly dependent on the nature of the γ-substituents.

The active site region of the vitamin K-dependent carboxylase includes both the amino-terminal hydrophobic and carboxy-terminal hydrophilic domains of the protein

In order to localize the active site of the vitamin K‐dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino‐terminal and hydrophilic carboxy‐terminal domains of the carboxylase, in contrast with previous work which localized both the catalytic and the propeptide binding site within the amino‐terminal hydrophobic domain. Amino acid analysis revealed that the mass of an amino‐terminal fragment is seriously underestimated by SDS‐PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy‐terminal hydrophilic domain.

Biological properties of selenobiotin

Abstract Selenobiotin is an excellent growth factor, as efficient as biotin in supporting the growth of biotin requiring microorganisms. It is incorporated in carboxylases leading to active “selenocarboxylases”. With E. Coli, the activity of the selenoacetyl CoA carboxylase is very similar to that of the normal enzyme.

Melanin biosynthesis: a study of polyphenol deoxygenation

The 1,3,6,8-tetrahydroxynaphthalene (T4HN) reductase of Verticillium dahliae has been studied in a cell-free system. The use of specifically labelled 4(R)- and [4(S)-2H] NADPH in the reduction of T4HN to scytalone reveals that the label is specifically transferred in the case of [4(S)-2H] NADPH whereas no deuterium transfer occurs with the 4R-isomer. This establishes that the T4HN reductase of V. dahliae is a NADPH-dependent dehydrogenase and that it belongs to class B.

Homolytic decarboxylation of glutamate analogues

Abstract The homolytic decarboxylation of a 3-cyclopropylglutamate derivative yields some rearrangement product whereas that of a 3-fluoroglutamate derivative yields the decarboxylation product without elimination. These results are discussed in relationship with the study of vitamin K-dependent carboxylation.

Synthesis of DL-5,5′-dihydroxyleucine the reduction product of γ carboxy glutamic acid

Abstract The synthesis of DL-5,5′-dihydroxyleucine, by diborane reduction of N-phaloyl-DL-γ-carboxyglutamic acid-α-methylester, and the chromatographic and spectral characteristics of this amino acid are reported.