Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases

Abstract

Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.

Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T

“…Up to 2 mg of PhNah20A was purified in two chromatographic steps from one liter of E. coli BL21(DE3) culture (see Section 3.4). Expression of truncated PhNah20A and PhNah20B, containing only the catalytic and not the GH20b domain (see Figure S3), did not result in protein production which is in agreement with previous findings that GH20b is essential for enzyme production and activity [50]. Attempts to produce PhNah20B without the CHB_HEX domains ( Figure S3) also gave no detected protein or β-NAHA activity.…”

“…PhNah20A contains two domains, the GH20 catalytic (β/α) 8 -barrel domain (Pfam: PF00728) and the N-terminal GH20b domain (also referred to as GH20 domain 2; Pfam: PF02838) of a predicted zincin-like fold similar to zinc-dependent metalloproteases [49] consisting of four antiparallel β-strands and an α-helix [27,50]. These two domains are typical for GH20 enzymes [50], and importantly they constitute an active and stable minimum functional unit of GH20 enzymes, thus requiring both a catalytic GH20 and a GH20b domain [50]. PhNah20A has no predicted signal peptide sequence and most probably is not secreted, whereas a 28 residues N-terminal signal peptide was predicted for the hypothetical PhNah20B ( Figure 1A).…”

“…PhNah20B, in addition to the GH20b and GH20 domains, contains a putative carbohydrate binding domain of the CHB_HEX superfamily (Pfam: PF03173) having a predicted β-sandwich structure similar to cellulose binding domains in cellulases [51], and a C-terminal CHB_HEX_C domain (Pfam: PF03174) of unknown function resembling an immunoglobulin-like fold [50,51]. A similar four-domain architecture was seen in the crystal structure of a chitobiase from S. marcescens [51], and has only been reported for bacterial GH20 enzymes [50,51]. Based on its protein sequence identity and domain architecture, PhNah20B resembles a biochemically uncharacterized GH20 chitobiase from Aliiglaciecola lipolytica and β-NAHAs from other phylogenetically close marine bacteria (Table S1).…”

A Second β-Hexosaminidase Encoded in the Streptococcus pneumoniae Genome Provides an Expanded Biochemical Ability to Degrade Host Glycans

“…Domain III is all ␣-helical with four anti-parallel helices organized in a near planar arrangement. Domain III is unusual among structurally characterized GH20 enzymes and has only been observed in GcnA (30,43). Indeed, GH20C and GcnA share the same domain organization and an overall root mean square deviation of 0.79 Å over 619 matched C␣s.…”

“…Indeed, GH20C and GcnA share the same domain organization and an overall root mean square deviation of 0.79 Å over 619 matched C␣s. Being the core catalytic domain of the GH20s, domain II is well conserved among all of the structurally characterized members of the family, whereas domain I is present in all but Aggregatibacter actinomycetemcomitans DspB and the two GH20s found in StrH (43,44). In these latter enzymes domain I is replaced by a FIVAR-like (found in various architectural regions) domain (9).…”