Structural and Enzymatic Analysis of Soybean β-Amylase Mutants with Increased pH Optimum

Abstract

Comparison of the architecture around the active site of soybean ␤-amylase and Bacillus cereus ␤-amylase showed that the hydrogen bond networks (Glu 380 -(Lys 295 -Met 51 ) and Glu 380 -Asn 340 -Glu 178 ) in soybean ␤-amylase around the base catalytic residue, Glu 380 , seem to contribute to the lower pH optimum of soybean ␤-amylase. To convert the pH optimum of soybean ␤-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean ␤-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0 -6.6). The K m values of the mutants were almost the same as that of soybean ␤-amylase except in the case of M51T, while the V max values of all mutants were low compared with that of soybean ␤-amylase. The crystal structure analysis of the wild typemaltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu 380 and Asn 340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu 380 and Lys 295 was also disrupted. These results indicated that the reduced pK a value of Glu 380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean ␤-amylase compared with that of the bacterial ␤-amylase.

Redox Regulation of a Novel Plastid-Targeted β-Amylase of Arabidopsis

“…Strong pH dependency and acidic pH optimum are common features among b-amylases (Hirata et al, 2004). Indeed, TR-BAMY, when assayed with the artificial substrate PNPG5, showed a narrow pH-activity curve with a definite maximum at pH 6.0 to 6.5 and dramatically reduced activity above neutrality.…”

“…pH-dependency of TR-BAMY activity. Purified recombinant TR-BAMY was assayed in Britton-Robinson buffer (Hirata et al, 2004) with either PNPG5 (white circles) or soluble starch (black circles). Data points are means 6 SD of triplicate determinations.…”

“…If not otherwise stated, all assays were performed at pH 6.0. The pH dependency of purified TR-BAMY was determined in Britton-Robinson buffer (Hirata et al, 2004).…”

Nucleotide sequence variation associated with .BETA.-amylase deficiency in the sweet potato Ipomoea batatas (L.) Lam

“…The movement of the inner loop ( 343 NFTCLEM 349 : corresponding to 343-349 in sweet potato) was reported to play critical roles in the catalytic mechanism and multiple-attack action of the enzyme in soybean (Kang et al 2005). Asn343 has important roles to the hydrogen-bond network, whose members are functionally linked to the catalytic residue and maintain active-site geometry in soybean (Fang and Ford 1998, Hirata et al 2004, Joshi et al 2000. From the study in soybean, Phe344, Thr345, and Cys346 are responsible for substrate binding and enzymatic activity control (Totsuka et al 1994, Pujadas andPalau 1997).…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…For example, in soybean -amylase, the hydrogen bond networks around the catalytic base residue (E380) of the enzyme were removed by point mutations, raising the optimal pH from 5.4 to the more neutral pH range of between 6 and 6.6 (Hirata et al, 2004a;Hirata et al, 2004b).…”