Identification and Signature Sequences of Bacterial Δ4,5Hexuronate-2-O-Sulfatases

Abstract

Glycosaminoglycan (GAG) sulfatases, which catalyze the hydrolysis of sulfate esters from GAGs, belong to a large and conserved sulfatase family. Bacterial GAG sulfatases are essential in the process of sulfur cycling and are useful for the structural analysis of GAGs. Only a few GAG-specific sulfatases have been studied in detail and reported to date. Herein, the GAG-degrading Photobacterium sp. FC615 was isolated from marine sediment, and a novel Δ 4,5 hexuronate-2- O -sulfatase (PB2SF) was identified from this bacterium. PB2SF specifically removed 2- O -sulfate from the unsaturated hexuronate residue located at the non-reducing end of GAG oligosaccharides produced by GAG lyases. A structural model of PB2SF was constructed through a homology-modeling method. Six conserved amino acids around the active site were chosen for further analysis using site-directed mutagenesis. N113A, K141A, K141H, H143A, H143K, H205A, and H205K mutants exhibited only feeble activity, while the H310A, H310K, and D52A mutants were totally inactive, indicating that these conserved residues, particularly Asp52 and His310, were essential in the catalytic mechanism. Furthermore, bioinformatic analysis revealed that GAG sulfatases with specific degradative properties clustered together in the neighbor-joining phylogenetic tree. Based on this finding, 60 Δ 4,5 hexuronate-2- O -sulfatases were predicted in the NCBI protein database, and one with relatively low identity to PB2SF was characterized to confirm our prediction. Moreover, the signature sequences of bacterial Δ 4,5 hexuronate-2- O -sulfatases were identified. With the reported signature motifs, the sulfatase sequence of the Δ 4,5 hexuronate-2- O -sulfatase family could be simply identified before cloning. Taken together, the results of this study should aid in the identification and further application of novel GAG sulfatases.

Biochemical characteristics and synergistic effect of two novel alginate lyases from Photobacterium sp. FC615

“…Marine bacterium Photobacterium sp. FC615 [33] was collected from Jiaozhou Bay, near Qingdao city in Shandong province, China. E. coli BL21 (DE3) was used for gene expression and it was cultured at 37 °C in Luria–Bertani (LB) broth or on LB broth agar (LB broth supplemented with 1.5% agar) with ampicillin (100 μg/ml).…”

“…In this study, two novel alginate lyases, AlyPB1 and AlyPB2, were identified from the draft genome of a marine bacterium, Photobacterium sp. FC615 [33]. AlyPB1 and AlyPB2 were characterized as endolytic and exolytic alginate lyases, respectively, and showed a strong synergistic effect on the complete digestion of alginate.…”

Research and Application of Chondroitin Sulfate/Dermatan Sulfate-Degrading Enzymes

“…Compared with exosulfatases, endosulfatases should be more useful enzymatic tools for CS/DS studies but seem to be very rare in nature. Our recent study has shown that CS/DS sulfatases with similar specificities have common signature sequences and can cluster to form a single branch in the phylogenetic tree, although they descended from separate ancestral genes (Wang et al, 2019a). Based on this finding, a series of 4,5 HexA-2-O-sulfatases have been successfully predicted and verified from sequences in GenBank (Wang et al, 2019a), and we believe that some CS/DS endosulfatases could also be found by using this method.…”

“…Technically, sulfatases with specific activity that allow them to selectively remove sulfate groups from CS/DS chains should be another important tool for the structural and functional studies of CS/DS. Based on the positions of sulfate groups in CS/DS chains, there are several types of specific sulfatases in animals and bacteria, such as N-acetylgalactosamine-4-O-sulfatase (Yamagata et al, 1968;Sugahara and Kojima, 1996;Wang et al, 2019b;Baum et al, 1959) and N-acetylgalactosamine-6-O-sulfatase (Yamagata et al, 1968;Sugahara and Kojima, 1996;Lim and Horwitz, 1981;Singh et al, 1976), which specifically hydrolyze sulfate groups on the C-4 and C-6 positions of GalNAc residues, respectively, and hexuronate-2-O-sulfatase, which specifically removes C-2 sulfate groups from saturated or unsaturated hexuronic acids derived from the digestion of CS/DS by GAG lyases (Sugahara and Kojima, 1996;Myette et al, 2003;Wang et al, 2019a; Tables 2, 3). Based on the sequence similarities, the GAG sulfatases were recently classified in the database SulfAtlas (http://abims.sb-roscoff.fr/sulfatlas/) ( Barbeyron et al, 2016).…”

“…Three 4,5 HexA-2-O-sulfatases have been identified from Flavobacterium heparinum, Bacteroides thetaiotaomicron and Photobacterium sp. FC615, that can specifically remove 2-O-sulfate ester from a HexA residue located at the nonreducing terminus of an unsaturated oligosaccharide (Sugahara and Kojima, 1996;Myette et al, 2003;Ulmer et al, 2014;Wang et al, 2019a). Two N-acetylgalactosamine-4-O-sulfatases from Proteus vulgaris and Photobacterium sp.…”