Draft Genome Sequence of Nonlabens ulvanivorans, an Ulvan-Degrading Bacterium

Journal of Biological Chemistry

Belnik

et al. 2016

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Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to D-glucuronic acid (GlcUA), L-iduronic acid (IdoUA), or D-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a ␤-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan lyases from three Alteromonadales isolated bacteria. Two homologous ulvan lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan lyases and therefore are the first representatives of a new family of polysaccharide lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and 1 H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently.

“…LTR, and Pseudoalteromonas sp. PLSV, (8,12,13). Genome annotation was manually confirmed by CAZy 4 and revealed that these strains encoded many polysaccharide-degrading enzymes.…”

Section: Table 2 Primers Used In This Studymentioning

confidence: 99%

mentioning

confidence: 99%

New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order

Kopel

Journal of Biological Chemistry

Belnik

et al. 2016

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“…For example, the protein found in the bacterium Nonlabens ulvanivorans, combines two catalytic modules, one attributed to a sulfatase, and the other to a glycoside hydrolase belonging to family GH78 (rhamnosidases). This organization reflects a genomic rearrangement adapted to the degradation of ulvan, the sulfated cell-wall matrix polysaccharide found in green algae of the genera Ulva and Enteromorpha (Kopel et al, 2014). Ulvan, composed of 3O-sulfate-rhamnose, is likely more efficiently degraded by a bi-modular protein.…”

Section: Modular Sulfatasesmentioning

confidence: 99%

“…(B) Ulva degradation PUL observed in Nonlabens ulvanivorans isolated from the feces of Aplysia sp. (Kopel et al, 2014). (C) Carrageenan PUL in Cellulophaga algicola.…”

Section: Biochemically Characterized Marine Polysaccharide Sulfatasesmentioning

confidence: 99%

Marine Polysaccharide Sulfatases

2017

Front. Mar. Sci.

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Polysaccharides are the most abundant and the most complex organic molecules in the ocean. In contrast to land polysaccharides, many marine polysaccharides are highly sulfated; in particular, the cell walls of macroalgae harbor a high diversity of sulfated polysaccharides (carrageenans, agarans, fucoidans, ulvans, etc.). These sulfated polysaccharides, biosynthesized by macroalgal primary producers, represent an important food source for heterotrophic organisms. Their biodegradation requires a set of enzymes that can cleave the glycosidic linkages of the carbohydrate backbone (called glycoside hydrolases) and the sulfate ester groups (called polysaccharide sulfatases). This review first provides on overview of the current state of knowledge on the classification and mechanisms of sulfatases in general. Then, based on an exploration of marine genomic and metagenomics data that reveals the diversity of carbohydrate sulfatases, it focuses on strategies to predict these sulfatases. In particular, the modularity of sulfatases and their location in marine polysaccharide utilization loci (PUL) provide clues as to their potential substrates and can drive future functional assays. Finally, the review underscores the low number of currently biochemically characterized marine carbohydrate sulfatases (e.g., agarases, carrageenanases, and fucose sulfatases). Bottlenecks encountered in studies on sulfatases likely lie in the difficulties in purifying them and producing them in heterologous systems.

“…Marine bacteria producing enzymes able to degrade ulvan polysaccharides were only recently discovered (10) and the first ulvan lyase from Nonlabens (Persicivirga) ulvanivorans strain PLR was biochemically characterized as an endolytic ulvan lyase (11). The genome of N. ulvanivorans was recently sequenced (12), followed by sequencing the genomes of Pseudoalteromonas sp. Strain PLSV (13) and Alteromonas sp.…”

mentioning

confidence: 99%

Structure–function analyses of a PL24 family ulvan lyase reveal key features and suggest its catalytic mechanism

Ulaganathan

Journal of Biological Chemistry

Kopel

et al. 2018

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Ulvan is a major cell wall component of green algae of the genus Ulva and some marine bacteria encode enzymes that can degrade this polysaccharide. The first ulvan degrading lyases have been recently characterized and several putative ulvan lyases have been recombinantly expressed, confirmed as ulvan lyases and partially characterized. Two families of ulvan degrading lyases, PL24 and PL25, have recently been established. The PL24 lyase LOR_107 from the bacterial Alteromonadales sp. strain LOR degrades ulvan endolytically, cleaving the bond at the C4 of a glucuronic acid. However, the mechanism and LOR_107 structural features involved are unknown. We present here the crystal structure of LOR_107, representing the first PL24 family structure. We found that LOR_107 adopts a seven-bladed β-propeller fold with a deep canyon on one side of the protein. Comparative sequence analysis revealed a cluster of conserved residues within this canyon, and site-directed mutagenesis disclosed several residues essential for catalysis. We also found that LOR_107 uses the His/Tyr catalytic mechanism, common to several PL families. We captured a tetrasaccharide substrate in the structures of two inactive mutants, which indicated a two-step binding event, with the first substrate interaction near the top of the canyon coordinated by Arg-320, followed by sliding of the substrate into the canyon toward the active-site residues. Surprisingly, the LOR_107 structure was very similar to that of PL25 family PLSV_3936, despite only ~14% sequence identity between the two enzymes. On the basis of our structural and mutational analyses, we propose a catalytic mechanism for LOR_107 that differs from the typical His/Tyr mechanism.Ulvan is one of the two major cell wall components of marine green algae (genus Ulva and Enteromorpha). It is a complex sulfated polysaccharide composed mainly of 3-sulfated rhamnose (Rha3S), glucuronic acid (GlcA), iduronic acid (IdoA) and xylose (1). The common disaccharide repetitive units within the ulvan polysaccharides are [→4)-β-D-GlcA-(1→4)-α-LRha3S-(1→] called type A ulvanobiourinic-3-sulfate (A 3S ) and [→4)-α-L-IdoA-(1→4)-α-LRha3S(1→] called type B ulvanobiouronic-3-sulfate (B 3S ) (1) (Schema 1). The presence of iduronic acid and sulfated rhamnose differentiates ulvan from other polysaccharides of marine origin and displays similarity with mammalian glycosaminoglycans such as chondroitin sulfate and hyaluronic acid. This distinctive chemical feature makes ulvan an attractive candidate for various biomedical, nanobiotechnological and drug delivery applications (2-6).Polysaccharides containing uronic acid sugars can be degraded by enzymes utilizing a β-http://www.jbc.org/cgi/doi/10.1074/jbc.RA117. (PLs). They utilize a β-elimination mechanism to cleave the oxygenaglycone bond by abstracting the C5 proton, which results in the formation of an unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (ΔUA) at the non-reducing end of the oligosaccharide product (7). These enzymes are presently classified into 26 s...