An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum

Dagmar Röther, L. Poppe, Gaby Morlock, Sandra Viergutz, János Rétey

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Abstract

The plant enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia-lyase (HAL) whose structure has been solved by X-ray crystallography. Based on amino-acid sequence alignment of the two enzymes, mutagenesis was performed on amino-acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino-acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in kcat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345-, 615- and 14-fold lower kcat, respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild-type PAL. To elucidate the possible function of the mutated amino-acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.

Original languageEnglish
Pages (from-to)3065-3075
Number of pages11
JournalEuropean Journal of Biochemistry
Volume269
Issue number12
DOIs
Publication statusPublished - 2002

Fingerprint

Histidine Ammonia-Lyase
Petroselinum
Phenylalanine Ammonia-Lyase
Mutagenesis
Catalytic Domain
Amino Acids
Catalysis
Enzymes
Sequence Alignment
X ray crystallography
X Ray Crystallography
Amino Acid Sequence
Kinetics
Substrates
Experiments

Keywords

  • Homology model
  • MIO
  • PAL
  • Phenylalanine ammonia-lyase
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry

Cite this

An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum. / Röther, Dagmar; Poppe, L.; Morlock, Gaby; Viergutz, Sandra; Rétey, János.

In: European Journal of Biochemistry, Vol. 269, No. 12, 2002, p. 3065-3075.

Research output: Contribution to journalArticle

Röther, Dagmar ; Poppe, L. ; Morlock, Gaby ; Viergutz, Sandra ; Rétey, János. / An active site homology model of phenylalanine ammonia-lyase from Petroselinum crispum. In: European Journal of Biochemistry. 2002 ; Vol. 269, No. 12. pp. 3065-3075.
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