Identification, characterization and functional expression of a tyrosine ammonia-lyase and its mutants from the photosynthetic bacterium Rhodobacter sphaeroides.
A tyrosine ammonia-lyase (TAL) enzyme from the photosynthetic bacterium Rhodobacter sphaeroides (RsTAL) was identified, cloned and functionally expressed in Escherichia coli, where conversion of tyrosine to p-hydroxycinnamic acid (pHCA) was demonstrated. The RsTAL enzyme is implicated in production of pHCA, which serves as the cofactor for synthesis of the photoactive yellow protein (PYP) in photosynthetic bacteria. The wild type RsTAL enzyme, while accepting both tyrosine and phenylalanine as substrate, prefers tyrosine, but a serendipitous RsTAL mutant identified during PCR amplification of the RsTAL gene, demonstrates much higher preference for phenylalanine as substrate and deaminates it to produces cinnamic acid. Sequence analysis showed the presence of three mutations: Met4 --> Ile, Ile325 --> Val and Val409 --> Met in this mutant. Sequence comparison with Rhodobacter capsulatus TAL (RcTAL) shows that Val409 is conserved between RcTAL and RsTAL. Two single mutants of RsTAL, Val409 --> Met and Val 409 --> Ile, generated by site-directed mutagenesis, demonstrate greater preference for phenylalanine compared to the wild type enzyme. Our studies illustrate that relatively minor changes in the primary structure of an ammonia-lyase enzyme can significantly affect its substrate specificity.
Xue
Zhixiong
Z
Biochemical Sciences and Engineering, DuPont CR&D, DuPont Experimental Station, Wilmington, DE 19880-0173, USA.
McCluskey
Michael
M
Cantera
Keith
K
Sariaslani
F Sima
FS
Huang
Lixuan
L
D000595:Amino Acid Sequence; D000642:Ammonia-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D020283:Photoreceptors, Microbial / Q000378:metabolism*; D010788:Photosynthesis; D011422:Propionates; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*
D001426:Bacterial Proteins; D003373:Coumaric Acids; D020283:Photoreceptors, Microbial; D011422:Propionates; C050162:photoactive yellow protein, Bacteria; D014443:Tyrosine; D000642:Ammonia-Lyases; C023208:L-tyrosine ammonia-lyase; C495469:p-coumaric acid