Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

FP5

DECARBOXYLATE Sintesi della relazione

Project ID: QLK1-CT-2002-02388
Finanziato nell'ambito di: FP5-LIFE QUALITY
Paese: Netherlands

Characterization of amino acid / biogenic amine exchangers

Biogenic amines (BA) are a major cause of food poisoning. Their appearance in food 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 food and beverages. BA are the end product of metabolic pathways that provide the microorganisms with metabolic energy, in the form of either ATP or proton motive force. Pathways producing proton motive force are simple pathways involving the decarboxylation of amino acids yielding the BA.

The ATP producing pathways are more complicated and involve deimination of guanidine groups. Both types of pathways involve transport proteins embedded in the cytoplasmic membrane, which are responsible for both the uptake of the precursor and the excretion of the BA in the cell. We have expressed three of these so-called precursor/product exchangers in a heterologous expression system and have functionally characterised them.

The three transporters are:
- The histidine/histamine exchanger HdcP of Lactobacillus hilgardii 0006;
- The tyorisine/tyramine exchanger TyrP of Lactobacillus brevis;
- The agmatine and putrescine exchanger AgmP of Lactobacillus brevis.

The transporters were expressed in the lactic acid bacterium Lactococcus lactis under control of the inducible nisin promoter. The structural genes coding for the transporters were cloned in vector pNZ8048 yielding plasmids pNZhdcP, pNZtyrP and pNZagmP, which encodes the exchangers with an additional N-terminal His-tag that was added for expression studies. The plasmids were transformed to L. lactis strain NZ9000 and the expressed proteins characterised in resting cells or membrane vesicles prepared from the cells.

The histidine transporter HdcP was identified as a membrane protein using antibodies raised against the N-terminal His-tag. It showed up in the membrane as a single protein with a apparent molecular mass of 40 kDa on SDS-PAGE (calculated molecular weight 55 kDa). L. lacis cells expressing HdcP had a twelve fold higher uptake activity of histidine relative to the control cells. Chase experiments demonstrated the ability of the transporter to catalyse homologous histidine/histidine exchange and heterologous histidine histamine exchange. The hdcP gene of L. hilgardii is the first gene that was experimentally shown to encode for a histidine/histamine exchanger.

The tyrosine transporter TyrP was identified as a protein with an apparent molecular weight of 44 kDa on SDS-PAGE (calculated molecular weight 54 kDa). Tyrosine uptake in cells of L. lactis expressing TyrP was stimulated 15-fold relative to cells lacking TyrP. Internalised tyrosine could be chased with excess tyrosine and with tyramine demonstrating the tyrosine/tyramine exchange capacity of TyrP. Studies with membrane vesicles with a right-side-out orientation demonstrated that TyrP had a low uniporter activity transporting tyrosine or tyramine in the absence of cotransport. The exchange activity between tyrosine and tyramine, which is very efficient, was shown to be electrogenic.

The agmatine transporter AgmP was expressed in L. lactis as a protein with an apparent molecular weight of 35 kDa (theoretical molecular weight of 53 kDa). L. lactis cells expressing AgmP catalysed uptake of agmatine and putrescine to high levels, while the control cells were devoid of this activity. The recombinant cells showed no higher uptake activity for arginine or ornithine demonstrating that the agmatine deiminase pathway and the arginine deiminase pathway make use of distinct transporters. The uptake was strongly dependent on the membrane potential component of the proton motive force suggesting that the transporter catalyses electrogenic uniport of the divalent, positively charged substrates. Agmatine/putrescine exchange was demonstrated by chasing accumulated putrescine with agmatine and vice versa.

The properties of the exchangers HdcP, TyrP and AgmP are those typical for precursor/product exchangers. They catalyse efficient exchange between two structurally related compounds. HdcP and TyrP operate in decarboxylation pathways that generate proton motive force and, therefore, catalyse electrogenic exchange. AgmP operates in a deiminase pathway that generates ATP and, therefore, catalyses electroneutral exchange. The transporters have in common that they catalyse unidirectional transport of the substrates at a much lower activity.

The studies provide a framework to scientist in the LAB scientific community to identify and characterise other transporters in LAB that are potential BA producers.

Informazioni correlate

Contatto

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