title: Structure modeling and functional analysis of recombinant dextransucrase from Weissella confusa Cab3 expressed in Lactococcus lactis.
abstract: The dextransucrase gene from Weissella confusa Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in Lactococcus lactis. The recombinant dextransucrase, WcCab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. WcCab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. WcCab3-rDSR gave Kmof 6.2 mM and Vmof 6.3 µmol/min/mg. The characterization of dextran synthesized by WcCab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of WcCab3-rDSR using the crystal structures of dextransucrase from Lactobacillus reuteri (protein data bank, PDB id: 3HZ3) and Streptococcus mutans (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (β/α)8triose phosphate isomerase-barrel fold making the catalytic core of WcCab3-rDSR. The structure superposition and multiple sequence alignment analyses of WcCab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.
authors: a Department of Biosciences and Bioengineering , Indian Institute of Technology Guwahati , Guwahati , Assam , India.
authors: Shraddha
authors: S
authors: Shukla
authors: a Department of Biosciences and Bioengineering , Indian Institute of Technology Guwahati , Guwahati , Assam , India.
authors: Anil Kumar
authors: AK
authors: Verma
authors: b VTT Technical Research Centre of Finland , Espoo , Finland.
authors: Ilkka
authors: I
authors: Kajala
authors: b VTT Technical Research Centre of Finland , Espoo , Finland.
authors: Antti
authors: A
authors: Nyyssolä
authors: a Department of Biosciences and Bioengineering , Indian Institute of Technology Guwahati , Guwahati , Assam , India.
authors: Rwivoo
authors: R
authors: Baruah
authors: b VTT Technical Research Centre of Finland , Espoo , Finland.|c Department of Food and Environmental Sciences , University of Helsinki , Helsinki , Finland.
authors: Kati
authors: K
authors: Katina
authors: b VTT Technical Research Centre of Finland , Espoo , Finland.
authors: Riikka
authors: R
authors: Juvonen
authors: c Department of Food and Environmental Sciences , University of Helsinki , Helsinki , Finland.
authors: Maija
authors: M
authors: Tenkanen
authors: a Department of Biosciences and Bioengineering , Indian Institute of Technology Guwahati , Guwahati , Assam , India.
authors: Arun
authors: A
authors: Goyal
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003911:Dextrans / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D058836:Weissella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D003911:Dextrans; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase
keywords: Dextran; Lactococcus lactis; NMR; Weissella confuse; dextransucrase; homology modeling