TY - JOUR
T1 - Initiation, Elongation, and Termination of Bacterial Cellulose Synthesis
AU - McManus, John B.
AU - Yang, Hui
AU - Wilson, Liza
AU - Kubicki, James D.
AU - Tien, Ming
N1 - Funding Information:
We thank Jochen Zimmer for his kind gift of the BcsA-BcsB expression plasmid and the Penn State fermentation facility for the growth of the AcsA-AcsB expressing cells. This work was supported as a part of the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under award no. DE-SC0001090.
Publisher Copyright:
© 2018 American Chemical Society.
PY - 2018/3/31
Y1 - 2018/3/31
N2 - Cellulose is the major component of the plant cell wall and composed of β-linked glucose units. Use of cellulose is greatly impacted by its physical properties, which are dominated by the number of individual cellulose strand within each fiber and the average length of each strand. Our work described herein provides a complete mechanism for cellulose synthase accounting for its processivity and mechanism of initiation. Using ionic liquids and gel permeation chromatography, we obtain kinetic constants for initiation, elongation, and termination (release of the cellulose strand from the enzyme) for two bacterial cellulose synthases (Gluconacetobacter hansenii and Rhodobacter sphaeroides). Our results show that initiation of synthesis is primer-independent. After initiation, the enzyme undergoes multiple cycles of elongation until the strand is released. The rate of elongation is much faster than that of steady-state turnover. Elongation requires cyclic addition of glucose (from uridine diphosphate-glucose) and then strand translocation by one glucose unit. Translocations greater than one glucose unit result in termination requiring reinitiation. The rate of the strand release, relative to the rate of elongation, determines the processivity of the enzyme. This mechanism and the measured rate constants were supported by kinetic simulation. With the experimentally determined rate constants, we are able to simulate steady-state kinetics and mimic the size distribution of the product. Thus, our results provide for the first time a mechanism for cellulose synthase that accounts for initiation, elongation, and termination.
AB - Cellulose is the major component of the plant cell wall and composed of β-linked glucose units. Use of cellulose is greatly impacted by its physical properties, which are dominated by the number of individual cellulose strand within each fiber and the average length of each strand. Our work described herein provides a complete mechanism for cellulose synthase accounting for its processivity and mechanism of initiation. Using ionic liquids and gel permeation chromatography, we obtain kinetic constants for initiation, elongation, and termination (release of the cellulose strand from the enzyme) for two bacterial cellulose synthases (Gluconacetobacter hansenii and Rhodobacter sphaeroides). Our results show that initiation of synthesis is primer-independent. After initiation, the enzyme undergoes multiple cycles of elongation until the strand is released. The rate of elongation is much faster than that of steady-state turnover. Elongation requires cyclic addition of glucose (from uridine diphosphate-glucose) and then strand translocation by one glucose unit. Translocations greater than one glucose unit result in termination requiring reinitiation. The rate of the strand release, relative to the rate of elongation, determines the processivity of the enzyme. This mechanism and the measured rate constants were supported by kinetic simulation. With the experimentally determined rate constants, we are able to simulate steady-state kinetics and mimic the size distribution of the product. Thus, our results provide for the first time a mechanism for cellulose synthase that accounts for initiation, elongation, and termination.
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U2 - 10.1021/acsomega.7b01808
DO - 10.1021/acsomega.7b01808
M3 - Article
AN - SCOPUS:85043316615
SN - 2470-1343
VL - 3
SP - 2690
EP - 2698
JO - ACS Omega
JF - ACS Omega
IS - 3
ER -