Document Detail


Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase.
MedLine Citation:
PMID:  18346767     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Tyrosine ammonia lyase (TAL) catalyzes the conversion of L-tyrosine to p-coumaric acid using a 3,5-dihydro-5-methylidene-4H-imidazole-4-one (MIO) prosthetic group. In bacteria, TAL is used for production of the photoactive yellow protein chromophore and for caffeic acid biosynthesis in certain actinomycetes. Here we biochemically examine wild-type and mutant forms of TAL from Rhodobacter sphaeroides (RsTAL). Kinetic analysis of RsTAL shows that the enzyme displays a 90-fold preference for L-tyrosine versus L-phenylalanine as a substrate. The pH-dependence of TAL activity with L-tyrosine and L-phenylalanine demonstrates a common protonation state for catalysis, but indicates a difference in charge-state for binding of either amino acid. Site-directed mutagenesis demonstrates that Ser150, Tyr60, and Tyr300 are essential for catalysis. Mutation of Ser150 to an alanine abrogates formation of the MIO prosthetic group, as shown by mass spectrometry, and prevents catalysis. The Y60F and Y300F mutants were inactive with both amino acid substrates, but bound p-coumaric and cinnamic acids with less than 12-fold changes in affinity compared the wild-type enzyme. Analysis of MIO-dithiothreitol adduct formation shows that the reactivity of the prosthetic group is not significantly altered by mutation of either Tyr60 or Tyr300. The mechanistic roles of Ser150, Tyr60, and Tyr300 are discussed in relation to the three-dimensional structure of RsTAL and related MIO-containing enzymes.
Authors:
Amy C Schroeder; Sangaralingam Kumaran; Leslie M Hicks; Rebecca E Cahoon; Coralie Halls; Oliver Yu; Joseph M Jez
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2008-03-17
Journal Detail:
Title:  Phytochemistry     Volume:  69     ISSN:  0031-9422     ISO Abbreviation:  Phytochemistry     Publication Date:  2008 May 
Date Detail:
Created Date:  2008-04-21     Completed Date:  2008-07-25     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0151434     Medline TA:  Phytochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1496-506     Citation Subset:  IM    
Affiliation:
The Donald Danforth Plant Science Center, St Louis, MO 63132, USA.
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MeSH Terms
Descriptor/Qualifier:
Ammonia-Lyases / genetics,  metabolism*
Bacterial Proteins / genetics,  metabolism*
Catalysis
Molecular Structure
Mutagenesis, Site-Directed
Protein Binding
Rhodobacter sphaeroides / enzymology,  genetics
Serine / chemistry,  genetics,  metabolism*
Spectrometry, Mass, Electrospray Ionization
Tyrosine / chemistry,  genetics,  metabolism*
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 55520-40-6/Tyrosine; 56-45-1/Serine; EC 4.3.1.-/Ammonia-Lyases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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