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FP5

DECARBOXYLATE Informe resumido

Project ID: QLK1-CT-2002-02388
Financiado con arreglo a: FP5-LIFE QUALITY
País: Netherlands

IInhibitors of tyrosine decarboxylation pathways

Biogenic amines (BA) are a major cause of food poisoning. Their appearance in foods is the result of unwanted microbiological activity. Understanding the metabolic pathways and enzymes involved in BA production by LAB is essential for the understanding of the mechanism behind the spoilage of foods and beverages. BA are the end product of metabolic pathways that provide the microorganisms with metabolic energy, mostly in the form of proton motive force. The pathways consist of a transport protein embedded in the cytoplasmic membrane and a metabolic enzyme that converts an amino acid into the corresponding BA by a decarboxylation reaction. The transporters are responsible for both the uptake of the precursor (the amino acid) and the excretion of the BA in the cell. The transporters are at the gate of the pathways and any unwanted activity of the fermenting organisms is best attacked there. We have screened a set of compounds for their ability to inhibit tyramine formation by Lactobacillus brevis by studying the substrate specificity of the tyorisine/tyramine exchanger TyrP.

The tyrosine/tyramine exchanger TyrP was expressed in the lactic acid bacteria Lactococcus lactis under control of the inducible nisin promoter. The structural genes coding for the transporter was cloned in vector pNZ8048 yielding plasmids pNZtyrP, which encode the exchangers with an additional N-terminal His-tag that was added for expression studies. The plasmids were transformed to L. lactis NZ9000 strain and the substrate specificity of TyrP was studied in membrane vesicles with a right-side-out orientation using heterologous exchange and inhibition studies.

BA are the direct decarboxylation product of amino acid, and, consequently, the transporters catalyse structurally related compounds. A set of 19 structural analogues of tyrosine modified at different parts of the molecule were assayed for their ability to compete with tyrosine in the TyrP transporter activities. The set included hydroxyphenyl propionic acid, hydroxyphenyl pyruvic acid, and hydroxyphenyl lactic acid there were modified at the C2 amino group, tyramine, L-tyrosinol, L-tyrosine methyl ester, and L-tyrosine hydrazide modifed at the C2 carboxylate group, D,L-a-methyl tyrosine modified at the C2 hydrogen atom, L-phenyl alanine, fluoro-L-phenyl alanine, chloro-L-phenylalanine,bromo-L-phenyl alanine, and iodo-L-phenyl alanine modifed at the para-hydroxyl group, and meta-DL-tyrosine, ortho-DL-tyrosine, and DL-dihydroxy-phenylalanine (DOPA) that are modified at the ring.

Modifications of the C2 carboxylate group that is removed in the decarboxylation reaction were well tolerated by TyrP. The kinetics with the two physiological substrates L-tyrosine and its decarboxylation product tyramine were comparable. Analysis of the substrate specificity of TyrP in the exchange reaction demonstrates that the amino group and the phenyl ring with the para hydroxyl group provide the most important interaction sites of the molecules with the protein. All modifications of these groups resulted in at least a 10-fold reduction of the exchange rate.

Remarkably, for many of the modifications the effect on the exchange rate was stronger than on the inhibition of counterflow, suggesting that translocation involves more critical interactions between substrate and protein than the initial binding step.

A potent inhibitor of tyramine production by the tyrosine decarboxylation pathway of Lactobacillus brevis should bind with high affinity to the transporter and, preferentially, should translocate the compound not or only slowly. Heterologous exchange assays predominantly for the ability to translocate, while the inhibition of counterflow assay measures affinity.

Therefore, a good inhibitor is poorly active in exchange and highly potent in inhibiting counterflow. With L-phenylalanine as the substrate, TyrP had a translocation activity that was far less than 10% under conditions where counterflow activity was inhibited by >95%. L-phenylalanine is one of the naturally occurring amino acids in foods. Adding additional L-phenyl alanine during the fermentations could prevent the formation of the BA tyramine.

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Contacto

Julius LOLKEMA, (Group leader)
Tel.: +31-50-3632155
Fax: +31-50-3632154
Correo electrónico
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