Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 β-Xylosidases

Abstract

The glycoside hydrolase family 39 (GH39) is a functionally expanding family with limited understanding about the molecular basis for substrate specificity and extremophilicity. In this work, we demonstrate the key role of the positive-subsite region in modulating substrate affinity and how the lack of a C-terminal extension impacts on oligomerization and structural stability of some GH39 members. The crystallographic and SAXS structures of a new GH39 member from the phytopathogen Xanthomonas citri support the importance of an extended C-terminal to promote oligomerization as a molecular strategy to enhance thermal stability. Comparative structural analysis along with sitedirected mutagenesis showed that two residues located at the positive-subsite region, Lys166 and Asp167, are critical to substrate affinity and catalytic performance, by inducing local changes in the active site for substrate binding. These findings expand the molecular understanding of the mechanisms involved in substrate recognition and structural stability of the GH39 family, which might be instrumental for biological insights, rational enzyme engineering and utilization in biorefineries.

Characterization of a highly xylose tolerant β-xylosidase isolated from high temperature horse manure compost

“…Modelling shows that His241, part of the β-hairpin catalytic loop and not present in other GH39 β-xylosidases, overlaps the position taken by Tyr283 possibly substituting for this residue. As noted elsewhere [ 27 , 28 ], overall, the catalytic site and substrate binding pockets are highly conserved, and these structures align very well. Thus, it should be expected that catalysis by XylP81 also operates by the catalytic mechanism (double displacement) described for GsXynB1 from G. stearothermophilus and TsXynB from Thermoanaerobacterium saccharolyticum [ 29 – 31 ].…”

“…The temperature optimum profile may therefore partly be the result of the substrate used, and we note that several GH39 β-xylosidases display broad temperature optima on pNPX. Inspection of the primary sequence showed that the C-terminal extension present in most GH39 β-xylosidases from extremophiles was not present [ 27 , 28 , 31 ]. This suggests that despite the overall sequence similarity to members of the Chloroflexi and other thermophilic genera, the enzyme likely derives from a mesophile.…”

“…This suggests that despite the overall sequence similarity to members of the Chloroflexi and other thermophilic genera, the enzyme likely derives from a mesophile. It also suggests that the enzyme is monomeric in solution as the C-terminal extension is thought to be the primary reason for tetramer formation in those enzymes which have this quaternary structure [ 28 , 34 ]. The pH optimum is 6, similar to the optima reported for other GH39 β-xylosidases (pH5—pH7.5) with a bell-shaped curve indicating two titratable groups at pKa values of ~ 4.6 and 7.4 [ 30 ].…”

“…Modelling of the XylP81 sequence on the structure of XacXynB (QMEAN − 3.80), and comparison of the XylP81 model with the structures of CcXynB2, TsXynB and GsXynB1 (Additional file 1 : Fig. S3) showed that, like CcXynB2 and XacXynB, XylP81 has the longer α-helix-containing loop from the auxiliary domain (Ser399-Glu419) that interacts with the catalytic β-hairpin forcing it to adopt an open conformation [ 28 , 34 ]. The K M for XylP81 on pNPX was 5.3 mM and is in line with what has been reported for most bacterial GH39 xylosidases (Figs.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…Rational design takes benefit of structures and sequences of already known stable proteins. Through identification of amino acids which are assumed to participate in (de)stabilization, new variants of proteins of interest are created through site-directed mutagenesis [79]. As an example, asparagine is a thermolabile residue prone to deamination which can be mutated into threonine or isoleucine, with similar geometry but more thermostable.…”