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Protein Engineering, Vol. 12, No. 8, 687-692, August 1999
© 1999 Oxford University Press

Analysis of stability and catalytic properties of two tryptophanases from a thermophile

Hiromi Kudo1, Ryo Natsume, Makoto Nishiyama2,3 and Sueharu Horinouchi

Department of Biotechnology and 2 Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Two tryptophanases, Tna1 and Tna2, both of which were cloned from the thermophile Symbiobacterium thermophilum, differ in their enzymatic properties, such as thermal stability, catalytic efficiency and activation energy of catalysis, despite the great similarity (92%) in their amino acid sequences. Chimeric tryptophanases were constructed by recombination of the two genes to try to elucidate the molecular basis for the difference. The stability of each chimeric enzyme was roughly proportional to the content of amino acid residues from Tna1. Three regions, tentatively named regions 2, 4 and 5, which contained the amino acid residues 70–129, 192–298 and 299–453, respectively, were especially important for the increase in thermal stability. Site-directed mutagenesis revealed that V104 in region 2 and Y198 in region 4 of Tna1 were involved in the increase in thermal stability of Tna1. Amino acid residues contributing to the higher catalytic efficiency of Tna1 were similarly analyzed, using the chimeric tryptophanases, and found to be located in region 5. Site-directed mutagenesis revealed that I383 and G395 in Tna1, which were presumably located close to the putative active center, played an active role in the increase of catalytic efficiency of Tna1. The activation energy of catalysis was proportional to the content of amino acid residues from Tna2, suggesting the amino acid residues responsible for the difference were dispersed over the whole molecule.

Keywords: activation energy/catalytic efficiency/thermal stability/thermostable enzyme/tryptophanase

1 Present address: Lead Generation Laboratory, Medical Research Laboratories, Taisho Pharmaceutical Co., Ltd, 403, Yoshino-cho 1-chome, Omiya-shi, Saitama, 330-8530, Japan

3 To whom correspondence should be addressed


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