Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in Taxol side-chain biosynthesis.
The conversion of alpha-phenylalanine to beta-phenylalanine is the first committed step in the biosynthesis of the C-13 side chain of Taxol. Thus, the novel enzyme responsible for this step, phenylalanine aminomutase (PAM), is of considerable interest for studies of Taxol biosynthesis and represents a potential target for genetic engineering. A method is described for purifying PAM from Taxus chinensis cell cultures. The purified enzyme has a K(m) of 1.1mM, a V(max) of 110.1 microm/min/mg protein, a pH optimum of 7.5-8.0, and a denatured molecular weight of about 80 kDa. Peptide sequences derived from the purified protein were used to design and synthesize degenerate primers enabling the PCR synthesis of the PAM cDNA. The PAM cDNA encodes a protein of 687 amino acid residues with a deduced molecular weight of 75.3 kDa. The PAM cDNA was cloned and expressed in Escherichia coli, and PAM activity was demonstrated. As a gene symbol for the PAM enzyme, pam is proposed. Protein sequence alignments of PAM, phenylalanine ammonia-lyase (PAL), and histidine ammonia-lyase (HAL) sequences exhibit significant similarity providing insight into potential active site residues of PAM.
Steele
Christopher L
CL
Fermentation and Biocatalysis Development, Technical Operations, Bristol-Myers Squibb Company, Syracuse, NY 13221-4755, USA. Csteele2@vrxus.jnj.com
Chen
Yijun
Y
Dougherty
Brian A
BA
Li
Wenying
W
Hofstead
Sandra
S
Lam
Kin S
KS
Xing
Zizhuo
Z
Chiang
Shu-Jen
SJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000096:biosynthesis*; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020946:Taxus / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase; D017239:Paclitaxel