Ishikawa Kohki, Mihara Yasuhiro, Shimba Nobuhisa, Ohtsu Naoko, Kawasaki Hisashi, Suzuki Ei-ichiro, Asano Yasuhisa
Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho Kawasaki-ku, Kawasaki 210-868, Japan.
Protein Eng. 2002 Jul;15(7):539-43. doi: 10.1093/protein/15.7.539.
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 K(m) value for inosine and thus increased the productivity of 5'-IMP. We determined the crystal structure of G74D/I153T, which has a reduced K(m) 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 K(m) value. Prompted by this hypothesis, we designed several mutations and introduced them to G74D/I153T, to decrease the K(m) value further. This strategy produced a S72F/G74D/I153T mutant with a 5.4-fold lower K(m) value and a 2.7-fold higher V(max) value as compared to the wild-type EB-NSAP.
蜚蠊埃希氏菌非特异性酸性磷酸酶（EB - NSAP）具有焦磷酸 - 核苷磷酸转移酶活性，该活性具有C - 5'位选择性。利用当前的突变和结构数据来构建一种突变型EB - NSAP，以作为一种有效且经济的蛋白质催化剂，用于从核苷合成核苷酸的潜在工业应用。首先，将Gly74和Ile153分别替换为Asp和Thr，因为摩根氏摩根菌同源酶中的相应替换降低了肌苷的K(m)值，从而提高了5'-肌苷酸（5'-IMP）的生产率。我们测定了G74D/I153T的晶体结构，正如预期的那样，其对肌苷的K(m)值降低。G74D/I153T的三级结构与野生型几乎相同。此外，尽管Asp74和Thr153这两个引入的侧链都位于潜在的肌苷结合位点附近，但在肌苷与EB - NSAP的假设结合模式中，它们都不直接参与与肌苷的相互作用。这些发现表明，潜在肌苷结合位点周围氨基酸替换引起的轻微结构变化可能会显著降低K(m)值。受此假设的启发，我们设计了几个突变并将其引入G74D/I153T，以进一步降低K(m)值。与野生型EB - NSAP相比，该策略产生了一种S72F/G74D/I153T突变体，其K(m)值低5.4倍，V(max)值高2.7倍。
