Initiation, Elongation, and Termination of Bacterial Cellulose Synthesis

Abstract

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.

Bionanocellulose – Properties, Acquisition And Perspectives Of Application In The Food Industry

“…Z tego powodu niektóre drobnoustroje wykształciły skomplikowany mechanizm służący produkcji nanobiomateriału celulozowego. Produkcja BC jest realizowana przez kompleks enzymatyczny -syntazę celulozową, która, zależnie od gatunku i warunków hodowli, wytwarza celulozę o odmiennych właściwościach fizykochemicznych [58].…”

Simulations of Cellulose Synthesis Initiation and Termination in Bacteria

“…The crystal structure of CESA was determined with the BcsA/BcsB complex from Rhodobacter sphaeroides. − This enzyme complex utilizes UDP-α- d -glucose as a substrate. , The BcsA subunit is the catalytically active subunit responsible for the formation of bacterial cellulose and translocation across the inner membrane . Within BcsA is the transmembrane (TM) tunnel from which the cellulose chain is extruded during the course of synthesis. ,, The chain length or degree of polymerization (DOP) of cellulose is a major determinant of the chemical and physical properties, with different species of bacteria able to generate cellulose with different lengths. − A high DOP can be desirable for the manufacture of paper and cotton cloth, whereas a low DOP may be desirable for the digestibility of biofuels . However, the structural components of CESAs and the molecular mechanism that controls the length of cellulose synthesized are not known.…”

“…Recently, our group (McManus et al) performed a kinetic analysis of BcsA/BcsB and proposed a mechanism accounting for the processivity and mechanism of initiation. The study involved a combination of kinetic and chemical analyses and kinetic simulations . McManus et al determined that the initiation of bacterial cellulose synthesis is primer-independent.…”

“…The study involved a combination of kinetic and chemical analyses and kinetic simulations . McManus et al determined that the initiation of bacterial cellulose synthesis is primer-independent. After initiation, the enzyme proceeds to an elongation phase that involves cyclic rounds of glucose addition from UDP-glucose to the nonreducing end of the chain followed by translocation of the chain by one glucose unit such that the active site is empty and is preparing the enzyme for the next elongation cycle .…”

Quantitative Characterization of the Amount and Length of (1,3)-β-D-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-D-glucan Synthase

“…SEC has also been applied to characterize bacterial cellulose synthase. In this case, cellulose was solubilized in dimethylacetamide containing 8% LiCl (w/v) and analyzed by gel permeation chromatography coupled to multi-angle light scattering (GPC-MALS) (McManus et al, 2018). This approach avoids the pitfalls of chemical derivatization and is potentially applicable to many other polysaccharides.…”