TY - JOUR
T1 - Crystal Structure of Levansucrase from the Gram-Negative Bacterium Brenneria Provides Insights into Its Product Size Specificity
AU - Xu, Wei
AU - Ni, Dawei
AU - Hou, Xiaodong
AU - Pijning, Tjaard
AU - Guskov, Albert
AU - Rao, Yijian
AU - Mu, Wanmeng
N1 - Funding Information:
We thank the staff of beamline BL18U1 of Shanghai Synchrotron Radiation Facility and Diamond Light Source (Oxfordshire, 0DE, UK) for assistance during X-ray data collection. We thank Lubbert Dijkhuizen for valuable insights and comments. This work was supported by the Natural Science Foundation of Jiangsu Province (No. BK20210463), and the National Natural Science Foundation of China (No. 31922073).
Publisher Copyright:
© 2022 American Chemical Society.
PY - 2022/4/27
Y1 - 2022/4/27
N2 - Microbial levansucrases (LSs, EC 2.4.1.10) have been widely studied for the synthesis of β-(2,6)-fructans (levan) from sucrose. LSs synthesize levan-type fructo-oligosaccharides, high-molecular-mass levan polymer or combinations of both. Here, we report crystal structures of LS from the G-bacterium Brenneria sp. EniD 312 (Brs-LS) in its apo form, as well as of two mutants (A154S, H327A) targeting positions known to affect LS reaction specificity. In addition, we report a structure of Brs-LS complexed with sucrose, the first crystal structure of a G-LS with a bound substrate. The overall structure of Brs-LS is similar to that of G- and G+-LSs, with the nucleophile (D68), transition stabilizer (D225), and a general acid/base (E309) in its active site. The H327A mutant lacks an essential interaction with glucosyl moieties of bound substrates in subsite +1, explaining the observed smaller products synthesized by this mutant. The A154S mutation affects the hydrogen-bond network around the transition stabilizing residue (D225) and the nucleophile (D68), and may affect the affinity of the enzyme for sucrose such that it becomes less effective in transfructosylation. Taken together, this study provides novel insights into the roles of structural elements and residues in the product specificity of LSs.
AB - Microbial levansucrases (LSs, EC 2.4.1.10) have been widely studied for the synthesis of β-(2,6)-fructans (levan) from sucrose. LSs synthesize levan-type fructo-oligosaccharides, high-molecular-mass levan polymer or combinations of both. Here, we report crystal structures of LS from the G-bacterium Brenneria sp. EniD 312 (Brs-LS) in its apo form, as well as of two mutants (A154S, H327A) targeting positions known to affect LS reaction specificity. In addition, we report a structure of Brs-LS complexed with sucrose, the first crystal structure of a G-LS with a bound substrate. The overall structure of Brs-LS is similar to that of G- and G+-LSs, with the nucleophile (D68), transition stabilizer (D225), and a general acid/base (E309) in its active site. The H327A mutant lacks an essential interaction with glucosyl moieties of bound substrates in subsite +1, explaining the observed smaller products synthesized by this mutant. The A154S mutation affects the hydrogen-bond network around the transition stabilizing residue (D225) and the nucleophile (D68), and may affect the affinity of the enzyme for sucrose such that it becomes less effective in transfructosylation. Taken together, this study provides novel insights into the roles of structural elements and residues in the product specificity of LSs.
KW - crystal structure
KW - fructooligosaccharides
KW - levan
KW - levansucrase
KW - product specificity
UR - http://www.scopus.com/inward/record.url?scp=85128373037&partnerID=8YFLogxK
U2 - 10.1021/acs.jafc.2c01225
DO - 10.1021/acs.jafc.2c01225
M3 - Article
C2 - 35388691
SN - 0021-8561
VL - 70
SP - 5095
EP - 5105
JO - Journal of Agricultural and Food Chemistry
JF - Journal of Agricultural and Food Chemistry
IS - 16
ER -