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Protein Engineering, Vol. 15, No. 7, 539-543, July 2002
© 2002 Oxford University Press

COMMUNICATION

Enhancement of nucleoside phosphorylation activity in an acid phosphatase

Kohki Ishikawa, Yasuhiro Mihara1, Nobuhisa Shimba, Naoko Ohtsu, Hisashi Kawasaki1, Ei-ichiro Suzuki2 and Yasuhisa Asano3

Central Research Laboratories and 1 Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho Kawasaki-ku, Kawasaki 210-8681 and 3 Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurosawa, Kosugi, Toyama 939-0398, Japan

Escherichia blattae non-specific acid phosphatase (EB-NSAP) possesses a pyrophosphate-nucleoside phosphotransferase activity, which is C-5'-position selective. Current mutational and structural data were used to generate a mutant EB-NSAP for a potential industrial application as an effective and economical protein catalyst in synthesizing nucleotides from nucleosides. First, Gly74 and Ile153 were replaced by Asp and Thr, respectively, since the corresponding replacements in the homologous enzyme from Morganella morganii reduced the Km value for inosine and thus increased the productivity of 5'-IMP. We determined the crystal structure of G74D/I153T, which has a reduced Km value for inosine, as expected. The tertiary structure of G74D/I153T was virtually identical to that of the wild-type. In addition, neither of the introduced side chains of Asp74 and Thr153 is directly involved in the interaction with inosine in a hypothetical binding mode of inosine to EB-NSAP, although both residues are situated near a potential inosine-binding site. These findings suggested that a slight structural change caused by an amino acid replacement around the potential inosine-binding site could significantly reduce the Km value. Prompted by this hypothesis, we designed several mutations and introduced them to G74D/I153T, to decrease the Km value further. This strategy produced a S72F/G74D/I153T mutant with a 5.4-fold lower Km value and a 2.7-fold higher Vmax value as compared to the wild-type EB-NSAP.


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