Substrate recognition by family 7 alginate lyase from Sphingomonas sp. A1

Ogura, Kohei; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

doi:10.1016/j.jmb.2008.05.008

Cited by 67 publications

(84 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3F), the distance between His 311 and C5 of Gϩ1 was calculated as 3.4 Å, and between Tyr 365 and O4 of Gϩ1 was less than 4.0 Å. Judging from the significant decrease in the specific activity of H311A and Y365F (Table 4), it is plausible that His 311 and Tyr 365 function as a catalytic base and acid, respectively (Fig. 5), as seen in A1-IIЈ (18). Tyr 555 is also a candidate for a catalytic acid because the distance between Tyr 555 and O4 of Gϩ1 was determined as 4.0 Å (Fig.…”

Section: Resultsmentioning

confidence: 95%

“…The crystal structures of PLs in 18 families have been determined, and the structure and functional relationships of enzymes such as pectin lyase in family PL-1 (14); pectate lyases in families PL-2, PL-3, and PL-10 (15-17); alginate lyases in families PL-5, PL-7, and PL-14 (13,18,19); rhamnogalacturonan lyases in families PL-4 and PL-11 (20,21); chondroitin lyases in families PL-6 and PL-8 (22); hyaluronan lyases in families PL-8 and PL-16 (23,24); heparin lyases in families PL-13 and PL-21 (25,26); and xanthan lyase in family PL-8 (12) have been demonstrated. On the other hand, no structural information on family PL-15 alginate lyases has been accumulated, and the enzyme mechanism for releasing unsaturated monosaccharides from the polysaccharide main chain remains to be clarified.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens

Ochiai

Yamasaki

Mikami

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a ␤-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-L-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/⌬GGG) were determined at 2.10-and 2.99-Å resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an ␣/␣-barrel ؉ anti-parallel ␤-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/⌬GGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His 311 and Tyr 365 as the catalytic base and acid, respectively. Two structural determinants, i.e. a short ␣-helix in the central ␣/␣-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.Carbohydrate-active enzymes such as glycoside hydrolases, polysaccharide lyases, glycosyl transferases, and carbohydrate esterases are categorized into more than 200 families based on amino acid sequences in the Carbohydrate-Active enZYmes (CAZy) data base (1). Polysaccharide lyases (PLs) 2 are classified into 21 PL families and they play an important role in degrading acidic polysaccharides such as polygalacturonan, rhamnogalacturonan, alginate, chondroitin, hyaluronan, heparin, heparan, and xanthan. These lyases commonly recognize uronic acid residues in polysaccharides, catalyze ␤-elimination, and produce unsaturated saccharides with CϭC double bonds at nonreducing terminal uronate residues.Alginate, produced by brown seaweeds as a major component of the cell wall matrix, is a linear polysaccharide composed of ␣-L-guluronate (G) and its C5 epimer ␤-D-mannuronate (M). Three blocks, such as poly-␣-L-guluronate (poly(G)), poly-␤-D-mannuronate (poly(M)), and heteropolymeric random sequences (poly(MG)), constitute the alginate polymer (2). The alginate degradation pathway has been characterized in some bacteria, virus, and abalone. In the bacterium Sphingomonas sp. strain A1, alginate is directly incorporated into the cytoplasm by the cell surface pit, periplasmic binding proteins, and ATPbinding cassette transporter. The incorporated alginate is subsequently depolymerized into unsaturated disaccharides, trisaccharides, and tetrasaccharides through the action of three cytoplasmic endotype alginate lyases, A1-I, A1-II, and A1-III (Fig. 1A) (3). The unsaturated oligosaccha...

show abstract

Section: Resultsmentioning

confidence: 95%

mentioning

confidence: 99%

Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens

Ochiai

Yamasaki

Mikami

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…In general, reduced polarity will increase the preference for M, which has a greater potential for hydrophobic interactions than G (44). This can be seen in Ser 37 (AlyA3) and Ser 86 (AlyA1), both of which show hydrogen bonding toward ϩ2 (Table 2). Substitution to Ile and Leu, respectively, therefore favors M at ϩ2, i.e.…”

Section: Discussionmentioning

confidence: 99%

“…Pro 39 terminates ␤-strand SA2 (Fig. 2), and P39T may lead to elongation of SA2, which in turn will distort the ␤-strand and affect hydrogen bonding by Ser 37 . Similar indirect effects can be seen for residues Thr 85 and possibly Thr 89 residing in loop L1.…”

Section: Discussionmentioning

confidence: 99%

“…A1 lyase A1-IIЈ (37) with GGG (Protein Data Bank code 2ZAB) and MMG (Protein Data Bank code 2ZAC) in the substrate binding pocket were downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (38). The structures were analyzed with ligand-protein contacts (39) and LigPlot (40) and visualized with SwissProtein Data BankViewer (41).…”

Section: Analysis Of Alginate Degradation By High Performance Anion Ementioning

confidence: 99%

See 1 more Smart Citation

Isolation of Mutant Alginate Lyases with Cleavage Specificity for Di-guluronic Acid Linkages

Tøndervik

Klinkenberg

Aarstad

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1-4-linked ␤-D-mannuronic acid (M) and ␣-L-guluronic acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a ␤-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ϳ7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca 2؉ , one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ϳ10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.

show abstract