A full-length cDNA encoding phenylalanine ammonia-lyase (PAL) from Zea mays L. was isolated and the coding region was expressed in Escherichia coli as a C-terminal fusion to glutathione S-transferase. After purification by glutathione-Sepharose chromatography, the glutathione S-transferase moiety was cleaved off and the resulting PAL enzyme analyzed. In contrast to PAL from dicots, this maize PAL isozyme catalyzed the deamination of both i-phenylalanine (PAL activity) and i-tyrosine (tyrosine ammonialyase activity). These results provide unequivocal proof that PAL and tyrosine ammonia-lyase activities reside in the same polypeptide. In spite of large differences in the Michaelis constant and turnover number of the two activities, their catalytic efficiencies are very similar. Also, both activities have the same p H and temperature optima. These results imply that maize can produce p-coumaric acid from both phenylalanine and tyrosine.PAL (EC 4.3.1.5) catalyzes the elimination of ammonia from L-Phe to yield (E)-cinnamic acid ( Fig. 1) (Koukol and Com, 1961), the first step in the phenylpropanoid pathway (for reviews, see Hahlbrock and Scheel, 1989;Dixon and Paiva, 1995;Whetten and Sederoff, 1995;Campbell and Sederoff, 1996). PAL activity has been found in some fungi and in a11 higher plants analyzed but not in animals. Because of the central function of PAL at a branch point of metabolism, this enzyme is one of the best studied in plants; the same is true for its corresponding gene(s).In a11 plants analyzed, PAL has been shown to occur as a tetramer, and multiple forms have often been isolated (Bolwell et al., 1985). PAL multigene families, such as those in tomato (Lycopersicon esculentum; Lee et al., 1992), parsley (Petroselinum crispum; Appert et al., 1994), or Arabidopsis thaliana (Wanner et al., 1995), explain at least in part these different forms. The occurrence of heteromers is likely but has as yet not been proven. On the other hand, apparent charge isoforms of a poplar PAL were recovered from insect cell cultures producing the protein from a baculovirus expression vector containing the respective cDNA (McKegney et al., 1996).Results from severa1 studies (Neish, 1961; Young et al., 1966;Havir et al., 1971;Jangaard, 1974) indicated that the enzyme from monocots utilizes Tyr (TAL activity) in addition to Phe, whereas the enzyme from dicots utilizes only Phe efficiently. The four homotetrameric parsley PAL (Havir et al., 1971), indicating that PAL from maize also has TAL activity. However, the uncertainty remains that a protein with TAL activity copurifies with PAL activity. Referring to the heterologous expression of parsley PAL cDNAs (Schulz et al., 1989; Appert et al., 1994), Whetten and Sederoff (1995) stated: "The same experiment could be performed with maize cDNA to test for PAL and TAL activities in the same polypeptide." This is exactly the experiment we report here. Expression of a PAL-specific cDNA from maize in Escherichia coli provided the ultimate proof that PAL and TAL activities reside in ...

cDNA clones for all enzymes of the prechorismate pathway of higher plants have previously been cloned, with the exception of the second enzyme of the pathway, 3-dehydroquinate synthase. Here we describe the isolation of a cDNA encoding a 3-dehydroquinate synthase from tomato which was identified by complementing a 3-dehydroquinate synthase-deficient Escherichia coli strain with a tomato cDNA library. The deduced amino acid sequence contains a putative N-terminal plastid-specific transit peptide, and the sequence of the mature enzyme resembles those of the corresponding bacterial enzymes more than of the fungal enzymes. Sequence identity was even higher between the tomato and E. coli sequences than between the E. coli and other known bacterial sequences. The abundance of 3-dehydroquinate synthase transcripts differ in the organs of tomato plants analyzed. In cultured tomato cells, the abundance of 3-dehydroquinate synthase transcripts increased 9-fold within 4 to 5 h of elicitor treatment.

No abstract

No abstract