Structural and Catalytic Properties of the Four Phenylalanine Ammonia‐Lyase Isoenzymes from Parsley (Petroselinum Crispum Nym.)

Abstract

Near-full-length cDNAs for the four phenylalanine ammonia-lyase (PAL) isoenzymes in parsley (Petruselinum crispum Nym.) were cloned and the complete amino acid sequences deduced. Fusion proteins with glutathione S-transferase were expressed in Escherichia coli, purified and cleaved. All of the resulting phenylalanine ammonia-lyase proteins, as well as the fusion proteins, were catalytically active. The turnover number of one selected isoenzyme, PAL-1, was estimated to be around 22 s-' for each active site. In contrast to a certain degree of differential expression in various parts of parsley plants, the four phenylalanine ammonia-lyase isoenzymes exhibited very similar apparent K,,, values for L-phenylalanine (15-24.5 pM) as well as identical temperature (58°C) and pH (8.5) optima. All of them were competitively inhibited by (E)-cinnamate with similar efficiency (K, values: 9

Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley.

“…This level was reduced to background activity upon partial mutation of box L (box Lm, TCTCAAAGCAA instead of TCTCACCTACC), a mutation which had previously been shown to strongly impair binding of nuclear proteins (17). The high basal activity level conferred by box L alone was further increased when the entire promoter region containing all three In parsley, the apparent lack of differential PAL1, PAL2, and PAL3 gene expression is paralleled by an almost total lack of enzyme-kinetic differences among all four PAL isoforms, even including PAL4 (18). This unexplained, apparent redundancy of PAL genes in parsley-and even more likely in tomato with at least 5 PAL genes (14) and potato with 40-50 PAL genes (10)-is particularly surprising in view of the existence of only two of the functionally closely related 4CL genes in both parsley (20) and potato (32).…”

“…First, PAL has been studied extensively in parsley (16,17) and has been shown to be encoded by a family of four distinct genes (6,7). Second, while the enzyme-kinetic properties of all four encoded isoforms were almost indistinguishable (18), preliminary results indicated a certain degree of differential gene expression (17,19). And finally, parsley is the only plant species for which…”

RNAi Suppression of Arogenate Dehydratase1 Reveals That Phenylalanine Is Synthesized Predominantly via the Arogenate Pathway in Petunia Petals

“…located downstream of the enzyme with the highest apparent K m for Phe was most sensitive to reduction in Phe level. The highest reduction was observed for phenylacetaldehyde, which is synthesized by PAAS, whose K m value (1.2 mM) is 17-to 80-fold higher than those of plant PALs (15 to 70 mM) (Hanson and Havir, 1981;Jorrin and Dixon, 1990;Appert et al, 1994;Cochrane et al, 2004;Kaminaga et al, 2006). By contrast, the least affected isoeugenol branch is located downstream of cinnamate 4-hydroxylase, which generally has a very low K m value toward cinnamic acid, ranging in plants from 2.5 to 9 mM (Gabriac et al, 1991;Koopmann et al, 1999;Hü bnera et al, 2003).…”

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

“…Several enzymes have previously been characterized both in vivo and in vitro, and the data are available from both patent and scientific literature (3,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). However, the conditions of kinetic analysis are often quite different and may not represent the extent to which the enzymes perform in vivo.…”