Crystal Structure and Evolution of a Prokaryotic Glucoamylase

Abstract

The first crystal structures of a two-domain, prokaryotic glucoamylase were determined to high resolution from the clostridial species Thermoanaerobacterium thermosaccharolyticum with and without acarbose. The Nterminal domain has 18 antiparallel strands arranged in β-sheets of a super-β-sandwich. The C-terminal domain is an (α/α) 6 barrel, lacking the peripheral subdomain of eukaryotic glucoamylases. Interdomain contacts are common to all prokaryotic Family GH15 proteins. Domains similar to those of prokaryotic glucoamylases in maltose phosphorylases (Family GH65) and glycoaminoglycan lyases (Family PL8) suggest evolution from a common ancestor. Eukaryotic glucoamylases may have evolved from prokaryotic glucoamylases by the substitution of the N-terminal domain with the peripheral subdomain and by the addition of a starch-binding domain.

Structural Similarity between a Starch-hydrolyzing Enzyme and an N-Glycan-Hydrolyzing Enzyme: Exohydrolases Cleaving α-1,X-Glucosidic Linkages to Produce β-Glucose

“…1C). Domains N and A form a super β-sandwich and an (α/α) 6 barrel, respectively, as observed in T. thermosaccharolyticum glucoamylase. Domains B and C are not found in the bacterial glucoamylase, and domain C is homologous with a surface layer homology (SLH) domain, which is reportedly necessary for the attachment of bacterial proteins to the cell wall (26).…”

“…A bacterial glucoamylase from Thermoanaerobacterium thermosaccharolyticum is shown in Fig. 1B (6). No starch-binding domain, as observed in fungal glucoamylases, is found at the C-terminal part, but rather, the enzyme is composed of an N-terminal β-sandwich domain, and an α-helical linker joins the N-terminal domain and the catalytic (α/α) 6 barrel domain.…”

“…1B (6). No starch-binding domain, as observed in fungal glucoamylases, is found at the C-terminal part, but rather, the enzyme is composed of an N-terminal β-sandwich domain, and an α-helical linker joins the N-terminal domain and the catalytic (α/α) 6 barrel domain. In addition to GH15, many of the other enzymes described in this paper share similar domain compositions.…”

“…The crystal structure of the catalytic domain of A. awamori glucoamylase, which was presented in 1992, was the first reported structure of enzymes in the families GH15, GH37, and GH63 (17). Fungal glucoamylases such as A. awamori glucoamylase are generally composed of 2 domains, an N-terminal catalytic domain consisting of an (α/α) 6 barrel fold and a C-terminal starch-binding domain, connected by an O-glycosylated linker (14,18). Initially, the 3-dimensional structures of the catalytic domain (17) and the starch-binding domain (19) determined separately, but recently, the intact structure of Hypocrea jecorina glucoamylase has been reported, as shown in Fig.…”

“…The enzymes are glucoamylase (EC 3.2.1.3; Fig. 1A,B) (5,6), glucodextranase (EC 3.2.1.70; Fig. 1C) (7), and trehalase (EC 3.2.1.28; Fig.…”