TY - JOUR
T1 - Reaction mechanism of prephenate dehydrogenase from the alternative tyrosine biosynthesis pathway in plants
AU - Holland, Cynthia K.
AU - Jez, Joseph M.
N1 - Funding Information:
This work was supported by the National Science Foundation (NSF; MCB-1614539 to J.M.J.). C.K.H. was supported by an NSF Graduate Research Fellowship (DGE-1143954). The authors thank Prof. Timothy Wencewicz for providing prephenate for use in these studies.
Publisher Copyright:
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2018/6
Y1 - 2018/6
N2 - Unlike metazoans, plants, bacteria, and fungi retain the enzymatic machinery necessary to synthesize the three aromatic amino acids l-phenylalanine, l-tyrosine, and l-tryptophan de novo. In legumes, such as soybean, alfalfa, and common bean, prephenate dehydrogenase (PDH) catalyzes the tyrosine-insensitive biosynthesis of 4-hydroxyphenylpyruvate, a precursor to tyrosine. The three-dimensional structure of soybean PDH1 was recently solved in complex with the NADP+ cofactor. This structure allowed for the identification of both the cofactorand ligand-binding sites. Here, we present steady-state kinetic analysis of twenty site-directed active-site mutants of soybean (Glycine max) PDH compared to wild-type. Molecular docking of the substrate, prephenate, into the active site of the enzyme revealed its potential interactions with the active site residues and made a case for the importance of each residue in substrate recognition and/or catalysis, most likely through transition state stabilization. Overall, these results suggested that the active site of the enzyme is highly sensitive to any changes, as even subtle alterations substantially reduced the catalytic efficiency of the enzyme.
AB - Unlike metazoans, plants, bacteria, and fungi retain the enzymatic machinery necessary to synthesize the three aromatic amino acids l-phenylalanine, l-tyrosine, and l-tryptophan de novo. In legumes, such as soybean, alfalfa, and common bean, prephenate dehydrogenase (PDH) catalyzes the tyrosine-insensitive biosynthesis of 4-hydroxyphenylpyruvate, a precursor to tyrosine. The three-dimensional structure of soybean PDH1 was recently solved in complex with the NADP+ cofactor. This structure allowed for the identification of both the cofactorand ligand-binding sites. Here, we present steady-state kinetic analysis of twenty site-directed active-site mutants of soybean (Glycine max) PDH compared to wild-type. Molecular docking of the substrate, prephenate, into the active site of the enzyme revealed its potential interactions with the active site residues and made a case for the importance of each residue in substrate recognition and/or catalysis, most likely through transition state stabilization. Overall, these results suggested that the active site of the enzyme is highly sensitive to any changes, as even subtle alterations substantially reduced the catalytic efficiency of the enzyme.
KW - Amino acids
KW - Biosynthesis
KW - Enzyme catalysis
KW - Glycine max
KW - Kinetics
UR - http://www.scopus.com/inward/record.url?scp=85083598969&partnerID=8YFLogxK
U2 - 10.1002/cbic.201800085
DO - 10.1002/cbic.201800085
M3 - Article
AN - SCOPUS:85083598969
SN - 1439-4227
VL - 19
SP - 1132
EP - 1136
JO - ChemBioChem
JF - ChemBioChem
IS - 11
ER -