Crystallization, molecular replacement solution, and refinement of tetrameric β‐amylase from sweet potato

Publisher Website
Google Scholar
Abstract
Sweet potato β‐amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P42212 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β‐amylase, with a crystal volume per protein mass (VM) of 2.57 Å3/Da and a solvent content of 52% by volume. The three‐dimensional structure of the tetrameric β‐amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R‐value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut‐off) with good stereochemistry. The subunit structure of sweet potato β‐amylase (crystallized in the absence of α‐cyclodextrin) is very similar to that of soybean β‐amylase (complexed with α‐cyclodextrin). The root‐mean‐square (RMS) difference for 487 equivalent Cα atoms of the two β‐amylases is 0.96 Å. Each subunit of sweet potato β‐amylase is composed of a large (α/β)8 core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C‐terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)8 barrel.

This publication has 37 references indexed in Scilit: