Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii

Phenylalanine ammonia-lyase (PAL, EC 4 3 1 5) from green bamboo was Isolated and cloned from the shell of Bambusa oldhamii The Km of bamboo shell PAL for (L)-Phe was 476 RM and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa indicating that PAL from bamboo also exists as a tetramer The optimum temperature for PAL activity was 50 degrees C and the optimal pH 90 The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing Four PAL genes designated as B0PAL1 to B0PAL4 were cloned from B oldhamii The open reading frames of B0PAL3 and B0PAL4 were 2142 and 2106 bp in size respectively B0PAL2-4 contained one intron and two exons but no intron was found in BoPAL1 B0PAL4 expressed in Escherichia cob possessed both PAL and tyrosine ammonia-lyase activities While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of B0PAL1 F133H and B0PAL2 F134H respectively showed decreased k(cat)/K-m values toward L-Phe whereas B0PAL2 F134H showed a slightly increased k(cat)/K-m value toward L-Tyr These data suggest other residues largely control Phe/Tyr substrate specificity An antibody raised against the purified shell PAL was generated for histochemical studies In bamboo shell and branch shoots PAL was localized primarily in sclerenchyma cells (C) 2010 Elsevier Ltd All rights reserved