Identification of Functionally Important Amino Acid Residues in Zymomonas mobilis Levansucrase

Yanase, Hideshi; Maeda, Masahiro; Hagiwara, Eigo; Yagi, Hiroyuki; Taniguchi, Kenji; Okamoto, Kenji

doi:10.1093/oxfordjournals.jbchem.a003258

Cited by 66 publications

(90 citation statements)

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“…Similarly, the D194N mutant enzyme nearly lost its hydrolysis activity, however, a small amount of highly polymerized fructan were produced, indicating that the transfructosylation activity remained intact. Further mutations of D194 (Glu, His, Gln, Ser, Ala) resulted in loss of sucrose hydrolysis activity which supports that D194 is essential for hydrolysis of sucrose-fructoside bond [13]. D194 and E278 have a direct role in the hydrolysis reaction and both are located in the central pocket, directly interacting with fructose ( Figure 4) like their counterparts in other bacterial levansucrases [12].…”

Section: Active Site Residues; D194 and E278 Are Located In Central Pmentioning

confidence: 62%

“…Mutation to a positively charged residue (E278H) had remarkable effects on sucrose hydrolysis activity, causing a 210-fold decrease in kcat value. Therefore, E278 is an essential amino acid for hydrolysis [13]. Similarly, the D194N mutant enzyme nearly lost its hydrolysis activity, however, a small amount of highly polymerized fructan were produced, indicating that the transfructosylation activity remained intact.…”

Section: Active Site Residues; D194 and E278 Are Located In Central Pmentioning

confidence: 99%

“…After a detailed investigation of mutagenesis studies in literature, we selected residues D194, E278, Cys121, Cys151, Cys244, W80, P340 and His296 as important amino acids for enzyme activity [13,24,25]. Since the structure of Z. mobilis levansucrase is not solved yet, specific locations of these amino acids are not revealed totally.…”

Section: Mapping Of Important Amino Acids On the Modelmentioning

confidence: 99%

“…These amino acids are D86, D247 and E342 for B. subtilis and according to sequence alignments, correspond to D48, D194 and E278 for Z. mobilis Lsc (Figure 2). Yanase et al, (2002) mutated these residues and showed that E278D had a 30-fold smaller kcat value for sucrose hydrolysis when compared with the wild type enzyme [13]. Mutation to a positively charged residue (E278H) had remarkable effects on sucrose hydrolysis activity, causing a 210-fold decrease in kcat value.…”

Section: Active Site Residues; D194 and E278 Are Located In Central Pmentioning

confidence: 99%

“…There are several studies investigating the Z. mobilis levansucrase product specificity based on amino acid mutagenesis approaches. Thus several amino acids were shown to be critical for levan or FOS production including His296, Cys244, Glu117 and P340 [13,24,25]. Yet the location of these amino acids is not known since the atomic structure of Z. mobilis levansucrase is not solved up to date.…”

Section: Introductionmentioning

confidence: 99%

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Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

Bakar¹,

Türköz²

2017

SDÜ Fen Bil Enst Der

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Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities. These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries. Levansucrases from Gram-positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram-negative bacteria preferentially produce fructooligosaccharides. Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters. In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure-function relationship of the enzyme. Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model. The structural model presents a five-bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases. Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket. Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure. The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme.

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Section: Active Site Residues; D194 and E278 Are Located In Central Pmentioning

confidence: 62%

Section: Active Site Residues; D194 and E278 Are Located In Central Pmentioning

confidence: 99%

Section: Mapping Of Important Amino Acids On the Modelmentioning

confidence: 99%

Section: Active Site Residues; D194 and E278 Are Located In Central Pmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

Bakar¹,

Türköz²

2017

SDÜ Fen Bil Enst Der

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Microbial and Enzymatic Polysaccharides

Kralj

Khandige

Guan

et al. 2020

Kirk-Othmer Encyclopedia of Chemical Technology

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Polysaccharides are ubiquitous and the largest substantive material class in nature (eg, structural in plants (cellulose, starch), algae (alginate), and (microbes) and throughout history polysaccharides have found extensive use in many industrial applications, for example in packaging (paper and board), fiber and textiles (eg, cotton and viscose), adhesives, and even in early engineered thermoplastic materials (eg, cellulose esters). Sourced from nature and in general biocompatible, many polysaccharides have applications across various food and food additive applications. The broad functionality and general good compatibility are attractive properties increasingly desired as focus on sustainability is increasing. This article will discuss the established product range of microbial polysaccharides utilized across a range of specialty applications such as food, personal care, and industrials. Product categories such as alginates, bacterial cellulose, curdlan, dextran, diutan, emulsan, fructan, gellan, pullulan, sceroglucan, succinoglycan, welan, and xanthan gum will be reviewed. In addition, the emerging platform technology enzymatic polymerization is discussed with special focus on the transformational promise for this approach toward engineered polysaccharide structures. This approach allows for the rational and systematic design of polysaccharide materials controlling aspects such as molecular weight, branching, and particle morphology.

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Mutational analysis of conserved regions harboring catalytic triad residues of the levansucrase protein encoded by the lsc‐3 gene (lsc3) of Pseudomonas syringae pv. tomato DC3000

Mardo

Visnapuu

Vija

et al. 2013

Biotech and App Biochem

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Levansucrase encoded by the lsc-3 (lsc3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, 18 single-amino-acid mutants of 13 positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219, and Glu303 were proved as members of the catalytic triad. Respective alanine replacement mutants were practically inactive with their k cat values reduced up to ∼130,000 times. Additionally, the requirements of Trp61, Gln301, and Arg304, located in conserved sequence blocks around the catalytic triad positions for the catalysis were shown. The catalytic significance of the position equivalent to Arg304 was shown for levansucrases for the first time. Replacement of Gln301 specifically affected the polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225, and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. Using 1 mg of Lsc3 protein, 15.4 g of FOS with a degree of polymerization from 3 to 7 can be synthesized in a 20 H reaction with 1,200 mM sucrose. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 L of induced culture of recombinant Escherichia coli. C 2013

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Identification of Functionally Important Amino Acid Residues in Zymomonas mobilis Levansucrase

Cited by 66 publications

References 0 publications

Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

Microbial and Enzymatic Polysaccharides

Mutational analysis of conserved regions harboring catalytic triad residues of the levansucrase protein encoded by the lsc‐3 gene (lsc3) of Pseudomonas syringae pv. tomato DC3000

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