Crystal structure of native α-L-rhamnosidase from Aspergillus terreus

Abstract

α‐l‐Rhamnosidases cleave terminal nonreducing α‐l‐rhamnosyl residues from many natural rhamnoglycosides. This makes them catalysts of interest for various biotechnological applications. The X‐ray structure of the GH78 family α‐l‐rhamnosidase from Aspergillus terreus has been determined at 1.38 Å resolution using the sulfur single‐wavelength anomalous dispersion phasing method. The protein was isolated from its natural source in the native glycosylated form, and the active site contained a glucose molecule, probably from the growth medium. In addition to its catalytic domain, the α‐l‐rhamnosidase from A. terreus contains four accessory domains of unknown function. The structural data suggest that two of these accessory domains, E and F, might play a role in stabilizing the aglycon portion of the bound substrate.

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site

“…However, several GH3 enzyme complexes with in crystallo-perfused Glc 10,11 or Glc-derivatives 12,13 are available. Additionally, the native GH78 α- l -rhamnosidase with a deep pocket-shaped active site also holds the entrapped Glc molecule, which was not perfused in crystals 14 .…”

An effective computational‐screening strategy for simultaneously improving both catalytic activity and thermostability of α‐l‐rhamnosidase

“…α‐ l ‐rhamnosidases, a glycoside hydrolase, exhibit a low sequence identity at only 20%–30%, but share a similar (α/α)6‐barrel catalysis domain and several β‐sandwiches (Cui et al, 2007; Fujimoto et al, 2013; Guillotin et al, 2019; O'Neill et al, 2015; Pachl et al, 2018). Six crystal structures (PDB: 2OKX, 3W5M, 3CIH, 4XHC, 6GSZ, and 6I60) from the GH78 family have been determined so far.…”

“…It is widely used in the debittering of citrus juices (Prakash et al, 2002; Yadav & Yadav, 2000) improving the aroma components of beverages (Caldini et al, 1994; Spagma et al, 2000). To date, only 29 α‐ l ‐rhamnosidases have been biochemically characterized, and six of them, namely, Bs RhaB (PDB entry 2OKX), Bt 1001 (PDB entry 3CIH), Sa Rha78A (PDB entry 3W5M), Ko Rha (PDB entry 4XHC), At Rha (PDB entry 6GSZ), and Dt Rha (PDB entry 6I60) have been illustrated in crystal structures in GH78 family (Cui et al, 2007; Fujimoto et al, 2013; Guillotin et al, 2019; O'Neill et al, 2015; Pachl et al, 2018). In previous studies, we cloned and expressed α‐ l ‐rhamnosidase (r‐Rha1) from Aspergillus niger JMU‐TS528 (Li et al, 2016) which belongs to the GH78 family in the CAZy database.…”

An effective computational-screening strategy for simultaneously improving both catalytic activity and thermostability of α-L-rhamnosidase

“…2.1 Compactional screening the mutant candidates with better catalysis efficiency and thermostability α-L-rhamnosidases, a glycoside hydrolase, exhibit a low sequence identity at only 20%-30%, but share a similar (α/α)6-barrel catalysis domain and several β-sandwiches. [36][37][38][39][40] Six crystal structures (PDB: 2OKX, 3W5M, 3CIH, 4XHC, 6GSZ and 6I60) from the GH78 family have been determined so far. By homology modelling with the crystal structures of α-L-rhamnosidases 2OKX, 3W5M, and 4XHC as templates, the structure of r-Rha1 was built and used for our research.…”

“…34,35 To date, only 29 α-L-rhamnosidases have been biochemically characterized, and six of them, namely Bs RhaB (PDB entry 2OKX), Bt 1001 (PDB entry 3CIH), Sa Rha78A (PDB entry 3W5M), Ko Rha (PDB entry 4XHC), At Rha (PDB entry 6GSZ), and Dt Rha (PDB entry 6I60) have been illustrated in crystal structures in GH78 family. [36][37][38][39][40] In previous studies, we cloned and expressed α-L-rhamnosidase (Rha1) from Aspergillus niger JMU-TS528, 41 which belongs to the GH78 family in the CAZy database. Simultaneously, we applied semi-conservative site-directed mutagenesis on the catalytic domain to increase the enzyme activity of Rha1.…”