Amino acid substitutions of His296 alter the catalytic properties of Zymomonas mobilis 10232 levansucrase.

Li, Shu Ying; Chen, Ming; Li, Gang; Yan, Yong Liang; Yu, Hai; Zhan, Yu; Peng, Zi Xin; Wang, Jin; Lin, Min

doi:10.18388/abp.2008_3217

Cited by 24 publications

(25 citation statements)

References 15 publications

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“…Similarly, in the GH68 family, the homologues R360 ( Bacillus subtilis levansucrase) and H296 ( Zymomonas mobilis levansucrase) are also known to greatly determine the transfructosylation properties of these enzymes (Chambert and Petit‐Glatron, 1991; Yanase et al. , 2002; Li et al. , 2008).…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of 6‐SST/6‐SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6‐kestose

et al. 2012

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SUMMARYFructans play important roles as reserve carbohydrates and stress protectants in plants, and additionally serve as prebiotics with emerging antioxidant properties. Various fructan types are synthesized by an array of plant fructosyltransferases belonging to family 32 of the glycoside hydrolases (GH32), clustering together with GH68 in Clan-J. Here, the 3D structure of a plant fructosyltransferase from a native source, the Pachysandra terminalis 6-SST/6-SFT (Pt6-SST/6-SFT), is reported. In addition to its 1-SST (1-kestose-forming) and hydrolytic side activities, the enzyme uses sucrose to create graminan-and levan-type fructans, which are probably associated with cold tolerance in this species. Furthermore, a Pt6-SST/6-SFT complex with 6-kestose was generated, representing a genuine acceptor binding modus at the +1, +2 and +3 subsites in the active site. The enzyme shows a unique configuration in the vicinity of its active site, including a unique D/Q couple located at the +1 subsite that plays a dual role in donor and acceptor substrate binding. Furthermore, it shows a unique orientation of some hydrophobic residues, probably contributing to its specific functionality. A model is presented showing formation of a b(2-6) fructosyl linkage on 6-kestose to create 6,6-nystose, a mechanism that differs from the creation of a b(2-1) fructosyl linkage on sucrose to produce 1-kestose. The structures shed light on the evolution of plant fructosyltransferases from their vacuolar invertase ancestors, and contribute to further understanding of the complex structure-function relationships within plant GH32 members.

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Section: Discussionmentioning

confidence: 99%

Crystal structure of 6‐SST/6‐SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6‐kestose

et al. 2012

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“…The amount of glucose produced in the inactivated reaction mixture was estimated by dinitrosalicylic acid colorimetry. 26 One unit of Ba-SacB activity was defined as the amount of enzyme required to produce 1 μmol of glucose per minute. The specific enzyme activity was calculated by dividing the enzyme activity by the protein content.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…A 0.5 mL mixture was then incubated at 40 °C for 30 min and inactivated by being boiled for 10 min. The amount of glucose produced in the inactivated reaction mixture was estimated by dinitrosalicylic acid colorimetry . One unit of Ba-SacB activity was defined as the amount of enzyme required to produce 1 μmol of glucose per minute.…”

Section: Methodsmentioning

confidence: 99%

Secretion of Bacillus amyloliquefaciens Levansucrase from Bacillus subtilis and Its Application in the Enzymatic Synthesis of Levan

Zhou

et al. 2021

ACS Food Sci. Technol.

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Levan is a polymer of fructose synthesized by levansucrase using sucrose as the substrate, which is employed as a viscosifier, stabilizer, emulsifier, and gelling or water-binding agent for diverse industrial applications. To enable a gentle and efficient purification process, we cloned the gene of levansucrase (sacB) from Bacillus amyloliquefaciens BH072 into an inducible vector harboring the signal peptide gene amyQ and then expressed it in Bacillus subtilis 168. The optimal pH values for d-glucose production and levan formation by the recombinant enzyme were 6.0 and 7.0, respectively, whereas the optimum temperatures were 40 and 35 °C, respectively. The K m and V max values for the recombinant levansucrase were calculated to be 0.71 mM sucrose and 0.49 μmol mL–1 min–1. Finally, levan biosynthesized by the recombinant enzyme was detected by thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR). TLC chromatograms of reaction products qualitatively indicated the formation of levan that was further assessed by high-performance liquid chromatography analysis. Some important functional groups and chemical bonds of the obtained dried levan powder were determined by FT-IR.

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“…These mutations resulted in a prominent increase in hydrolysis, low yields of transfructosylation products and inability to synthesize FOS larger than DP 4–5 10,18,21,26 . The equivalent mutation R296Q in Zymomonas mobilis levansucrase increased the amount of tri-, tetra- and pentasaccharides 27 .…”

Section: Resultsmentioning

confidence: 99%

Exploring the sequence variability of polymerization-involved residues in the production of levan- and inulin-type fructooligosaccharides with a levansucrase

Possiel

Ortiz-Soto

Ertl

et al. 2019

Sci Rep

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The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called “precision medicine”. Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family (Glycoside Hydrolase 68, GH68). F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.

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Amino acid substitutions of His296 alter the catalytic properties of Zymomonas mobilis 10232 levansucrase.

Cited by 24 publications

References 15 publications

Crystal structure of 6‐SST/6‐SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6‐kestose

Crystal structure of 6‐SST/6‐SFT from Pachysandra terminalis, a plant fructan biosynthesizing enzyme in complex with its acceptor substrate 6‐kestose

Secretion of Bacillus amyloliquefaciens Levansucrase from Bacillus subtilis and Its Application in the Enzymatic Synthesis of Levan

Exploring the sequence variability of polymerization-involved residues in the production of levan- and inulin-type fructooligosaccharides with a levansucrase

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