Crystal structure of thermophilic dextranase fromThermoanaerobacter pseudethanolicus

Abstract

The crystal structures of the wild type and catalytic mutant Asp-312→Gly in complex with isomaltohexaose of endo-1,6-dextranase from the thermophilic bacterium Thermoanaerobacter pseudethanolicus (TpDex), belonging to the glycoside hydrolase family 66, were determined. TpDex consists of three structural domains, a catalytic domain comprising an (β/α)8-barrel and two β-domains located at both N- and C-terminal ends. The isomaltohexaose-complex structure demonstrated that the isomaltohexaose molecule was bound across the catalytic site, showing that TpDex had six subsites (-4 to +2) in the catalytic cleft. Marked movement of the Trp-376 side-chain along with loop 6, which was the side wall component of the cleft at subsite +1, was observed to occupy subsite +1, indicating that it might expel the cleaved aglycone subsite after the hydrolysis reaction. Structural comparison with other mesophilic enzymes indicated that several structural features of TpDex, loop deletion, salt bridge and surface-exposed charged residue, may contribute to thermostability.

Isomaltooligosaccharide-binding structure of Paenibacillus sp. 598K cycloisomaltooligosaccharide glucanotransferase

“…According to the subsite nomenclature for GHs [21], the position of the reducing end glucose is designated as subsite –1, the position of the seven glucose moieties from the reducing end (Glc–1 to Glc–7) are designated as subsites –1 to –7. The four glucose moieties Glc–1 to Glc–4 were located along the catalytic cleft, and occupied four subsites –1 to –4, which is a common structural feature of other GH66-enzymes solved previously [9–11]. Subsites –4 to –7 were located between the catalytic and CBM35-1 domains.…”

“…GH66 dextranases produce only isomaltooligosaccharides (IGs), whereas CITases produce mainly CIs and small amounts of IGs. Currently, three 3D structures of GH66 enzymes have been determined and the enzymes have a modular protein architecture with a (β/α) 8 TIM-barrel as a GH66 catalytic domain and several other β-domains [9–11]. Three domains, the catalytic domain and two β-domains on the N- and C-terminal ends, are conserved in most GH66 enzymes, and all the known CITases have extra β-domains.…”

Structural diversification during glucosinolate breakdown: mechanisms of thiocyanate, epithionitrile and simple nitrile formation

“…Contrary to all other loop models, the four most promising 3L2 conformations showed C α, L151 to S aglucone sulfate distances similar to or smaller than those determined with the previously known TaTFP model (18.26 Å, Table ). The best evaluated 3L2 model was based on the template structure 5AXG:119–141 (Suzuki et al ., ) and had a C α, L151 to S aglucone sulfate distance of 13.04 Å.…”

Cytosolic N‐Glycans: Triggers for Ubiquitination Directing Proteasomal and Autophagic Degradation

“…[23] Although NGLY1 is a deglycosylation enzyme, it has additional domains involved in interactions with other proteins such as VCP and RAD23. [24] VCP is an ATPase that segregates ubiquitinated substrates from protein complexes or membranes, and its function is best understood in the ERAD pathway. [25] RAD23 contains ubiquitin-associated (UBA) domains for binding of ubiquitin chains and a ubiquitin-like (UBL) domain for proteasome association, and functions a shuttling factor between the VCP-NPL4-UFD1 complex and the proteasome.…”