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

Kopel, Moran; Helbert, William; Belnik, Yana; Buravenkov, Vitaliy; Herman, Asael; Banin, Ehud

doi:10.1074/jbc.m115.673947

Cited by 38 publications

(41 citation statements)

References 26 publications

(38 reference statements)

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“…The GlcA could be fitted in the +1 position in a chair conformation, while IdoA fitted only in a high energy boat conformation. In addition, since LOR_107 shows specificity for cleavage next to GlcA (15) we have modeled GlcA in the +1 position. The substrate is located close to, and makes contacts with, Arg320, Tyr330 and Asn263 (cluster II).…”

Section: Structure Of R259n and R320n Mutants With Ulvan Tetrasaccharidementioning

confidence: 99%

“…Our numbering follows the NCBI annotation. We have cloned residue 26-522 (without signal peptide) into the expression vector (pET28a) with C terminal 6xHis tag (15). LOR_107 gene includes several codons that are rare in Escherichia coli and for that reason we have used BL21-codon plus (DE3)-Ripl cells for transformation with the expression vector.…”

Section: Experimental Procedures Cloning and Purification Of Lor_107mentioning

confidence: 99%

“…The tetrasaccharides were obtained by enzymatic digestion of ulvan polysaccharide with LOR_107 ulvan lyase (PL24) from Alteromonas LOR sp. (15) and separated by HPLC chromatography. Tetrasaccharide I (DP4-1) contained a mixture of ΔUA-Rha3S-GlcARha3Sa/b and ΔUA-Rha3S-IdoA-Rha3Sa/b with some traces of hexasaccharides.…”

Section: Crystallization Of Lor_107 Mutants (R259n R320n and Y243f) mentioning

confidence: 99%

“…Initial characterization of LOR_107 showed that it preferentially cleaves the bond adjacent to glucuronic acid but has very low or no activity for ulvan segments containing iduronic acid (15). We report the crystal structure of LOR_107 at 1.9 Å resolution.…”

mentioning

confidence: 98%

“…strains LTR and LOR (14). Several of the putative ulvan lyase genes have been recombinantly expressed and partially characterized (15). LOR_107 from Alteromonas sp.…”

mentioning

confidence: 99%

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

Ulaganathan

Helbert

Kopel

et al. 2018

Journal of Biological Chemistry

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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...

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Section: Structure Of R259n and R320n Mutants With Ulvan Tetrasaccharidementioning

confidence: 99%

Section: Experimental Procedures Cloning and Purification Of Lor_107mentioning

confidence: 99%

Section: Crystallization Of Lor_107 Mutants (R259n R320n and Y243f) mentioning

confidence: 99%

mentioning

confidence: 98%

“…strains LTR and LOR (14). Several of the putative ulvan lyase genes have been recombinantly expressed and partially characterized (15). LOR_107 from Alteromonas sp.…”

mentioning

confidence: 99%

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

Ulaganathan

Helbert

Kopel

et al. 2018

Journal of Biological Chemistry

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Marine Polysaccharides: Occurrence, Enzymatic Degradation and Utilization

2021

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Macroalgae species are fast growing and their polysaccharides are already used as food ingredient due to their properties as hydrocolloids or they have potential high value bioactivity. The degradation of these valuable polysaccharides to access the sugar components has remained mostly unexplored so far. One reason is the high structural complexity of algal polysaccharides, but also the need for suitable enzyme cocktails to obtain oligo‐ and monosaccharides. Among them, there are several rare sugars with high value. Recently, considerable progress was made in the discovery of highly specific carbohydrate‐active enzymes able to decompose complex marine carbohydrates such as carrageenan, laminarin, agar, porphyran and ulvan. This minireview summarizes these achievements and highlights potential applications of the now accessible abundant renewable resource of marine polysaccharides.

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