The Arabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoforms.
In Arabidopsis thaliana, four genes have been annotated as provisionally encoding PAL. In this study, recombinant native AtPAL1, 2, and 4 were demonstrated to be catalytically competent for l-phenylalanine deamination, whereas AtPAL3, obtained as a N-terminal His-tagged protein, was of very low activity and only detectable at high substrate concentrations. All four PALs displayed similar pH optima, but not temperature optima; AtPAL3 had a lower temperature optimum than the other three isoforms. AtPAL1, 2 and 4 had similar K(m) values (64-71 microM) for l-Phe, with AtPAL2 apparently being slightly more catalytically efficacious due to decreased K(m) and higher k(cat) values, relative to the others. As anticipated, PAL activities with l-tyrosine were either low (AtPAL1, 2, and 4) or undetectable (AtPAL3), thereby establishing that l-Phe is the true physiological substrate. This detailed knowledge of the kinetic and functional properties of the various PAL isoforms now provides the necessary biochemical foundation required for the systematic investigation and dissection of the organization of the PAL metabolic network/gene circuitry involved in numerous aspects of phenylpropanoid metabolism in A. thaliana spanning various cell types, tissues and organs.
Cochrane
Fiona C
FC
Institute of Biological Chemistry, Washington State University, Clark Hall 467, Pullman, WA 99164-6340, USA.
Davin
Laurence B
LB
Lewis
Norman G
NG
D017360:Arabidopsis / Q000201:enzymology*; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D005810:Multigene Family; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D007527:Isoenzymes; D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase