The structure of PfGH50B, an agarase from the marine bacterium Pseudoalteromonas fuliginea PS47

Abstract

The recently identified marine bacterium Pseudoalteromonas fuliginea sp. PS47 possesses a polysaccharide-utilization locus dedicated to agarose degradation. In particular, it contains a gene (locus tag EU509_06755) encoding a β-agarase that belongs to glycoside hydrolase family 50 (GH50), PfGH50B. The 2.0 Å resolution X-ray crystal structure of PfGH50B reveals a rare complex multidomain fold that was found in two of the three previously determined GH50 structures. The structure comprises an N-terminal domain with a carbohydrate-binding module (CBM)-like fold fused to a C-terminal domain by a rigid linker. The CBM-like domain appears to function by extending the catalytic groove of the enzyme. Furthermore, the PfGH50B structure highlights key structural features in the mobile loops that may function to restrict the degree of polymerization of the neoagaro-oligosaccharide products and the enzyme processivity.

Keywords: agarase; agarose; glycoside hydrolase; marine bacterium; neoagaro-oligosaccharides.

Figure 1

Overall structure of PfGH50B. (a) Cartoon and (b) surface representation of PfGH50B. The N-terminal CBM-like domain is represented in pink, followed by a ∼30-amino-acid linker in yellow and the C-terminal catalytic domain in blue. The dashed line highlights the catalytic groove in (b). (c) Structural overlay of PfGH50B with SdAga50D (grey; PDB entry 4bq2) and AgWH50D (light brown; PDB entry 5z6p). The loops L1 and L2 in SdAga50D, which are disordered in PfGH50B, are highlighted in green.

Figure 2

Architecture of the PfGH50B catalytic groove. (a) PfGH50B (catalytic module in blue and CBM-like domain in pink) is overlaid with SdAga50D (green residues; PDB entry 4bq5) in complex with neoagaro-octaose (grey with subsites labelled in red). PfGH50B residues are numbered and SdAga50D residues are numbered in parentheses. Dashed lines and red spheres represent hydrogen bonds and water molecules, respectively. (b) Sequence alignment of PfGH50B loops L1 and L2 with the corresponding loops in SdAga50D (represented using ESPript 3.0; http://espript.ibcp.fr; Robert & Gouet, 2014 ▸). PfGH50B residue Asp352 (Asp362 in SdAga50D) in loop L1 is indicated by a black dot. (c) Surface representation of PfGH50B (catalytic module in blue and CBM-like domain in pink) with the missing loops L1 and L2 (in green) modelled based on the SdAga50D structure. The overlaid neoagaro-octaose from the SdAga50D complex structure (PDB entry 4bq5) is shown as grey sticks.