Comparison of Biochemical Characteristics, Action Models, and Enzymatic Mechanisms of a Novel Exolytic and Two Endolytic Lyases with Mannuronate Preference

Recent explorations of tool-like alginate lyases have been focused on their oligosaccharide-yielding properties and corresponding mechanisms, whereas most were reported as endo-type with α-L-guluronate (G) preference. Less is known about the β-D-mannuronate (M) preference, whose commercial production and enzyme application is limited. In this study, we elucidated Aly6 of Flammeovirga sp. strain MY04 as a novel M-preferred exolytic bifunctional lyase and compared it with AlgLs of Pseudomonas aeruginosa (Pae-AlgL) and Azotobacter vinelandii (Avi-AlgL), two typical M-specific endolytic lyases. This study demonstrated that the AlgL and heparinase_II_III modules play indispensable roles in determining the characteristics of the recombinant exo-type enzyme rAly6, which is preferred to degrade M-enriched substrates by continuously cleaving various monosaccharide units from the nonreducing end, thus yielding various size-defined ΔG-terminated oligosaccharides as intermediate products. By contrast, the endolytic enzymes Pae-rAlgL and Avi-rAlgL varied their action modes specifically against M-enriched substrates and finally degraded associated substrate chains into various size-defined oligosaccharides with a succession rule, changing from ΔM to ΔG-terminus when the product size increased. Furthermore, site-directed mutations and further protein structure tests indicated that H195NHSTW is an active, half-conserved, and essential enzyme motif. This study provided new insights into M-preferring lyases for novel resource discoveries, oligosaccharide preparations, and sequence determinations.

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Biochemical Characteristics and Variable Alginate-Degrading Modes of a Novel Bifunctional Endolytic Alginate Lyase

Applied and Environmental Microbiology ◽

10.1128/aem.01608-17 ◽

2017 ◽

Vol 83 (23) ◽

Cited By ~ 12

Author(s):

Yuanyuan Cheng ◽

Dandan Wang ◽

Jingyan Gu ◽

Junge Li ◽

Huihui Liu ◽

...

Keyword(s):

Alginate Lyase ◽

Fluorescent Labeling ◽

Structure Identification ◽

Key Factors ◽

Content Type ◽

Biochemical Characteristics ◽

Action Mode ◽

Molecular Sizes ◽

Sugar Chains ◽

Alginate Lyases

ABSTRACT Bifunctional alginate lyases can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G), but their substrate-degrading modes have not been thoroughly elucidated to date. In this study, we present Aly1 as a novel bifunctional endolytic alginate lyase of the genus Flammeovirga. The recombinant enzyme showed optimal activity at 50°C and pH 6.0. The enzyme produced unsaturated disaccharide (UDP2) and trisaccharide fractions as the final main alginate digests. Primary substrate preference tests and further structure identification of various size-defined final oligosaccharide products demonstrated that Aly1 is a bifunctional alginate lyase and prefers G to M. Tetrasaccharide-size fractions are the smallest substrates, and M, G, and UDP2 fractions are the minimal product types. Remarkably, Aly1 can vary its substrate-degrading modes in accordance with the terminus types, molecular sizes, and M/G contents of alginate substrates, producing a series of small size-defined saturated oligosaccharide products from the nonreducing ends of single or different saturated sugar chains and yielding unsaturated products in distinct but restricted patterns. The action mode changes can be partially inhibited by fluorescent labeling at the reducing ends of oligosaccharide substrates. Deletion of the noncatalytic region (NCR) of Aly1 caused weak changes of biochemical characteristics but increased the degradation proportions of small size-defined saturated M-enriched oligosaccharide substrates and unsaturated tetrasaccharide fractions without any size changes of degradable oligosaccharides, thereby enhancing the M preference and enzyme activity. Therefore, our results provided insight into the variable action mode of a novel bifunctional endolytic alginate lyase to inform accurate enzyme use. IMPORTANCE The elucidated endolytic alginate lyases usually degrade substrates into various size-defined unsaturated oligosaccharide products (≥UDP2), and exolytic enzymes yield primarily unsaturated monosaccharide products. However, it is poorly understood whether endolytic enzymes can produce monosaccharide product types when degrading alginate. In this study, we demonstrated that Aly1, a bifunctional alginate lyase of Flammeovirga sp. strain MY04, is endolytic and monosaccharide producing. Using various sugar chains as testing substrates, we also proved that key factors causing Aly1's action mode changes are the terminus types, molecular sizes, and M/G contents of substrates. Furthermore, the NCR fragment's effects on Aly1's biochemical characteristics and alginate-degrading modes and corresponding mechanisms were discovered by gene truncation and enzyme comparison. In summary, this study provides a novel bifunctional endolytic tool and a variable action mode for accurate use in alginate degradation.

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Structural and Biochemical Characteristics of Pathogenic Fungus: Cell Walls, Lipids and Dimorphism, and Action Modes of Antifungal Agents.

Nippon Ishinkin Gakkai Zasshi ◽

10.3314/jjmm.41.211 ◽

2000 ◽

Vol 41 (4) ◽

pp. 211-217 ◽

Cited By ~ 10

Author(s):

Yasuo Kitajima

Keyword(s):

Cell Walls ◽

Antifungal Agents ◽

Pathogenic Fungus ◽

Biochemical Characteristics ◽

Action Modes

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Characterization of Three New Azotobacter vinelandii Alginate Lyases, One of Which Is Involved in Cyst Germination

Journal of Bacteriology ◽

10.1128/jb.00455-09 ◽

2009 ◽

Vol 191 (15) ◽

pp. 4845-4853 ◽

Cited By ~ 44

Author(s):

Martin Gimmestad ◽

Helga Ertesvåg ◽

Tonje Marita Bjerkan Heggeset ◽

Olav Aarstad ◽

Britt Iren Glærum Svanem ◽

...

Keyword(s):

Azotobacter Vinelandii ◽

Alginate Lyase ◽

Growth Conditions ◽

Type I ◽

Wild Type ◽

Mannuronic Acid ◽

Guluronic Acid ◽

Polysaccharide Lyases ◽

Alginate Lyases

ABSTRACT Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic acid and α-l-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2+. All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

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A mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

10.1101/2021.03.31.437818 ◽

2021 ◽

Author(s):

Margrethe Gaardløs ◽

Tonje Marita Bjerkan Heggeset ◽

Anne Tøndervik ◽

David Tezé ◽

Birte Svensson ◽

...

Keyword(s):

Reaction Mechanism ◽

Azotobacter Vinelandii ◽

Alginate Lyase ◽

Cleavage Sites ◽

High Sequence Identity ◽

Preferential Cleavage ◽

Product Characterization ◽

Lyase Activity ◽

Mechanistic Basis ◽

Alginate Lyases

Mannuronan C-5 epimerases and alginate lyases are important enzymes for tailoring of the functional properties of alginate. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes, like AlgE7 from Azotobacter vinelandii, can perform both reactions. These enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their dual activity is not understood. In this study, we investigate mechanistic determinants involved in the bifunctional alginate lyase and epimerase activity of AlgE7. Based on sequence analyses a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Calcium promotes lyase activity whereas NaCl reduces the lyase activity of AlgE7. By using well-defined alginate substrates, the preferential cleavage sites of AlgE7 are found to be M|XM and G|XM. From the study of variants, it was found that R148 is particularly important for the lyase activity of AlgE7, and we obtained pure epimerase variants. Furthermore, the results suggest a catalytic reaction mechanism, with H154 as the catalytic base and Y149 as the catalytic acid. This study opens for further applications of alginate epimerases and lyases, by providing a better understanding of the reaction mechanism and how the two enzyme reactions can be altered by changes in reaction conditions.

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Comparison of the Values of a Novel Exolytic and Two Endolytic Alginate Lyases with Mannuronate Preference for Direct Preparation of Oligosaccharides, Action Modes, and Underlying Catalytic Mechanisms

10.21203/rs.3.rs-664516/v1 ◽

2021 ◽

Author(s):

Lianghuan Zeng ◽

Junge Li ◽

Yuanyuan Cheng ◽

Dandan Wang ◽

Jingyan Gu ◽

...

Keyword(s):

Fluorescent Labeling ◽

Specific Structure ◽

Direct Preparation ◽

Catalytic Mechanisms ◽

Action Modes ◽

Alginate Lyases ◽

Sequence Characteristics

Abstract Recent exploration of tool-like alginate lyases has focused on their oligosaccharide products and corresponding substrate action modes, and most were characterized as endolytic lyases with guluronate (G) preference. Herein, we elucidated a novel exolytic lyase, Aly-6, and two typical endolytic lyases, AlgL-Pae and AlgL-Avi, all with mannuronate (M) preferences. AlgL and heparinase_II_III modules play essential roles in determining the similar characteristics of these enzymes, although they are quite different in sequence characteristics. Aly-6 degraded substrates completely by continuously cleaving various monosaccharide units from nonreducing ends and producing various size-defined ΔG-terminated oligosaccharide fractions as intermediate alginate digests, which was inhibited by fluorescent labeling of reducing ends. Distinctly, AlgL-Pae and AlgL-Avi varied their action modes toward associated alginate substrates and therefore eventually degraded alginate into various size-defined oligosaccharide products with a specific structure-based succession rule. This study provided new insights into the action modes, associated mechanisms, and enzyme applications of M-preferred lyases.

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The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073313 ◽

1973 ◽

Vol 31 ◽

pp. 574-575

Author(s):

J. T. Stasny ◽

R. C. Burns ◽

R. W. F. Hardy

Keyword(s):

Cellular Localization ◽

Direct Evidence ◽

Azotobacter Vinelandii ◽

Intracellular Localization ◽

Protein Component ◽

Electron Microscopic ◽

Thin Sections ◽

Fe Protein ◽

Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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Microtubule Dynamics and Organelle Transport in Reticulomyxa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158510 ◽

1990 ◽

Vol 48 (3) ◽

pp. 194-195

Author(s):

Yih-Tai Chen ◽

Ursula Euteneuer ◽

Ken B. Johnson ◽

Michael P. Koonce ◽

Manfred Schliwa

Keyword(s):

Plasma Membrane ◽

Light Microscopy ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Microtubule Dynamics ◽

Model Systems ◽

Organelle Transport ◽

Biochemical Characteristics ◽

Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

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