Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida

Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3 ) from Pseudomonas putida by X-ray crystallography revealed that the electr ophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H -i-midazol-4-one (MIO) [Schwede, T.F., Retey, J., Schutz, G.E. (1999) Bioch emistry, 38, 5355-5361]. To evaluate the importance of several amino-acid r esidues at the active site for substrate binding and catalysis, we mutated the following amino-acid codons in the HAL gene: R283, Y53, Y280, E414, Q27 7, F329, N195 and H83. Kinetic measurements with the overexpressed mutants showed that all mutations resulted in a decrease of catalytic activity. The mutants R283I, R283K and N195A were approximate to 1640, 20 and 1000 times less active, respectively, compared to the single mutant C273A, into which all mutations were introduced. Mutants Y280F, F329A and Q277A exhibited ap proximate to 55, 100 and 125 times lower activity, respectively. The greate st loss of activity shown was in the HAL mutants Y53F, E414Q, H83L and E414 A, the last being more than 20 900-fold less active than the single mutant C273A, while H83L was 18 000-fold less active than mutant C273A. We propose that the carboxylate group of E414 plays an important role as a base in ca talysis. To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with L-cysteine and dioxygen at pH 10.5 as an indicator. All mutants , except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group. The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in H AL mutant F329A also. For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were pe rformed.