Characterization of a PA14 domain-containing galactofuranose-specific β-d-galactofuranosidase from Streptomyces sp.

Abstract

As a constituent of polysaccharides and glycoconjugates, β-d-galactofuranose (Galf) exists in several pathogenic microorganisms. Although we recently identified a β-d-galactofuranosidase (Galf-ase) gene, ORF1110, in the Streptomyces strain JHA19, very little is known about the Galf-ase gene. Here, we characterized a strain, named JHA26, in the culture supernatant of which exhibited Galf-ase activity for 4-nitrophenyl β-d-galactofuranoside (pNP-β-d-Galf) as a substrate. Draft genome sequencing of the JHA26 strain revealed a putative gene, termed ORF0643, that encodes Galf-ase containing a PA14 domain, which is thought to function in substrate recognition. The recombinant protein expressed in Escherichia coli showed the Galf-specific Galf-ase activity and also released galactose residue of the polysaccharide galactomannan prepared from Aspergillus fumigatus, suggesting that this enzyme is an exo-type Galf-ase. BLAST searches using the amino acid sequences of ORF0643 and ORF1110 Galf-ases revealed two types of Galf-ases in Actinobacteria, suggesting that Galf-specific Galf-ases may exhibit discrete substrate specificities.

Draft Genome Sequence of Streptomyces sp. JHA26, a Strain That Harbors a PA14 Domain Containing β- d -Galactofuranosidase

“…However, we found that the AbfB domain of ORF1110 was dispensable for the Gal f -ase activity of both substrates of artificial p NP-β- d -Gal f and natural Aspergillus fumigatus galactomannan ( 8 ). In contrast, we found that the PA14 domain of ORF0643 was important for the Gal f -ase activity of A. fumigatus galactomannan ( 9 ). We have also found that there are two types of Gal f -ases among the Actinobacteria : one type containing the AbfB domain and the other type containing the PA14 domain ( 9 ).…”

Galactofuranose-Related Enzymes: Challenges and Hopes

“…Another Galf -ase gene was also found later in the genome of Streptomyces sp., strain JHA26, coding for 869 amino acids. The Galf -ase specific enzyme was expressed, purified and characterized with the highest activity at pH 4.5 and temperature stability up to 45 • C and with K M of 6.8 mM for pNP-β-d-Galf as a substrate [65,66]. The most recent addition of cloned enzymes is the one from 2019, which provided the complete biochemical and kinetic characterization of subcloned Streptomyces spp.…”

Improvement of the versatility of an arabinofuranosidase against galactofuranose for the synthesis of galactofuranoconjugates

“…This enzymatic activity has been observed and monitored in various microorganism species for decades, [15][16][17][18] but proteins responsible for these activities were never isolated until recently. 19,20 Even so, the recently cloned galactofuranosidases have quite low activities (K M values of 4.4-6.8 mM, no reported k cat values) and have only been characterized in hydrolysis mode of either the natural Aspergillus fumigatus galactomannan or the non-natural substrate p-nitrophenyl-β-D-galactofuranoside 1 so their potential as a synthesis tool has not been demonstrated. Fortunately, β-D-galactofuranose and α-L-arabinofuranose are closely related glycosides, their only difference being an additional hydroxymethyl group on the C-5 of galactofuranose compared to arabinofuranose (Figure 1), so we supposed that enzymes acting on L-arabinofuranose might also have an activity against D-galactofuranose, or at least could be a starting point for the development of a biocatalyst using galactofuranose as substrate.…”