4.D.3 The Glycan Glucosyl Transferase (OpgH) Family
Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing E. coli cells are more than twice as large as their slow growing counterparts. Hill et al. (2013) reported the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. OpgH may sequester FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no detectable sequence similarity, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis revealed conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.
Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Morgan et al. 2013 presented the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate revealed the architecture of the cellulose synthase, demonstrated how BcsA forms a cellulose-conducting channel, and suggested a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.
Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. All catalytic subunits, known as cellulose synthase A (CESA) proteins, are S-acylated (Kumar et al. 2016). Analysis of Arabidopsis CESA7 revealed four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment.
Cellulose synthase is a membrane-integrated processive glycosyltransferase that couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Verma et al. 2023 presented substrate- and product-bound cryoEM structures of the homotrimeric cellulose synthase isoform-8 (CesA8) from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate's glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site revealed its critical function for catalytic activity. Molecular dynamics simulations revealed prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation (Verma et al. 2023).
References:
The glycan biosynthetic glucosyl transferase, OpgH (MdoH) of 847 aas and 6 - 9 TMSs.
Proteobacteria
OpgH of E. coli
Putative integral membrane glycosyl transferase, GT, of 451 aas and 4 TMSs.
GT of Chlamydomonas reinhardtii (Chlamydomonas smithii)
Putative polysaccharide synthase and transporter, NdvB, essential for normal biofilm formation (Zhang and Mah 2008).
NdvB of Pseudomonas aeruginosa
Cellulose synthase 2, bcsABII-A of 1518 aas and 10 TMSs.
bcsABII-A of Komagataeibacter xylinus (Gluconacetobacter xylinus)
Cellulose synthase catalytic subunit (UDP-forming) of 1550 aas and 10 TMSs.
Cellulose synthase of Komagataeibacter europaeus
Cellulose synthase, CesA7, of 1042 aas and 8 TMSs in a 2 +3 + 3 TMS arrangement where the first two TMSs are in the first half of the protein while the last 6 are C-terminal. The cryo-EM structure of the homotrimeric CesA7 from Gossypium hirsutum has been reported at 3.5 Å resolution (Zhang et al. 2021). The GhCesA7 homotrimer shows a C3 symmetrical assembly. Each protomer contains seven transmembrane helices (TMSs) which form a channel potentially facilitating the release of newly synthesized glucans. The cytoplasmic glycosyltransferase domain (GT domain) of GhCesA7 protrudes from the membrane, and its catalytic pocket is directed towards the TM pore. The homotrimer GhCesA7 is stabilized by transmembrane helix 7 (TMS 7) and the plant conserved region (PCR) domains. It represents the building block of CSCs and facilitates microfibril formation. This structure provides insight into how eukaryotic cellulose synthase assembles (Zhang et al. 2021).
Cellulose synthase, CesA7, of Gossypium hirsutum (Upland cotton) (Gossypium mexicanum)
Cellulose synthase of 516 aas and 6 - 8 TMSs.
Euryarchaea
Cellulose synthase of Methanoculleus bourgensis (Methanogenium bourgense)
Glycosyl transferase group 2 family of 523 aas and 7 TMSs.
Tenericutes
Glycosyl transferase of Mycoplasma bovis
Trimeric cellulose synthase coomplex for primary cell wall synthesis, CESA1, 3 and 6 (Watanabe et al. 2015). CESA1 is of 1084 aas and 8 TMSs while other CESA subunits are of similar size. All three are catalytic
subunit of the cellulose synthase complex. Required
for beta-1,4-glucan microfibril crystallization, a major mechanism of primary cell wall formation. Required during embryogenesis for cell elongation, orientation of cell
expansion and complex cell wall formation (Bashline et al. 2014).
determined the structure of a poplar cellulose synthase CesA homotrimer
that suggests a molecular basis for cellulose microfibril formation.
This complex, stabilized by cytosolic plant-conserved regions and
helical exchange within the transmembrane segments, forms three channels
occupied by nascent cellulose polymers. Secretion steers the polymers
toward a common exit point, which could facilitate protofibril
formation. CesA's N-terminal domains assemble into a cytosolic stalk
that interacts with a microtubule-tethering protein and may thus be
involved in CesA localization.
Plants
Cellulose synthase complex of Arabidopsis thaliana (Mouse-ear cress)
Cellulose synthase and transporter, BcsA, which functions with BcsB (periplasmic protein with an N-terminal TMS) and BcsC (an 18 β-stranded outer membrane porin). The x-ray structure of the BcsA-B complex has been determined at 3.5 Å resolution (Morgan et al. 2013). Cellulose is synthesized and secreted by the membrane-integrated cellulose synthase. Substrate- and product-bound structures of BcsA provided the basis for substrate recognition and demonstrated the stepwise elongation of cellulose. Structural snapshots showed that BcsA translocates cellulose via a ratcheting mechanism involving a 'finger helix' that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves 'up' and 'down' in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA's transmembrane channel. This mechanism was validated by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation (Morgan et al. 2016).
BcsA of Rhodobacter spheroides
Core cellulose synthase complex, BcsA/BcsB (Morgan et al. 2013; Omadjela et al. 2013). BcsA (YhjO) is an 10 - 12 TMS protein with 4 N-terminal TMSs, the glycosyl transferase domain and 4 C-terminal TMSs. It is believed to both synthesize the glycosyl linkages and transport the polysaccharide across the membrane (Römling and Galperin 2015). BcsB (YhjN) is a cellulose synthase regulator, a cyclic diGMP binding protein, with two TMSs at the N- and C-termini and the bulk of the protein in the periplasm. BcsC (TC# 1.B.55.3.1) is the outer membrane porin for the export of cellulose with an N-terminal α-TMS, a large N-terminal hydrophylic domain, and a C-terminal β-barrel domain. Two other proteins, an endoglucanase, BscZ or YhjM, and a diguanylate cyclase, YedQ (TC# 9.B.34.1.4) may play roles in cellulose biosynthesis (Imai et al. 2014). Cellulose is a component of the bacterial extracellular matrix (Zogaj et al. 2001) and plays roles in biofilm formation and cell adhesion (Hu et al. 2015).
BcsAB of E. coli
Cellulose synthase complex for secondary cell wall synthesis including CESA1, 3 and 6, all catalytic subunits (see 4.D.3.1.4) (Watanabe et al. 2015). A homolog (83% identical to Q8LPK5) from Populus tomentosa (Chinese white poplar) (CesA8) alone secretes the nascent polymer through a channel formed by its own transmembrane domain dependent on a lipid bilayer and Mn2+ to form microfibrils in vitro (Purushotham et al. 2016). CesA8, of 1055 aas and 6 - 8 TMSs, makes cellulose from UDP-activated glucose molecules. CesA8 is a membrane-integrated processive glycosyltransferase (Verma et al. 2023). It couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Verma et al. 2023 presented substrate and product-bound cryogenic EM structures of the homotrimeric CesA8 from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate's glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site reveals its critical function for catalytic activity. Molecular dynamics simulations revealed prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation. Molecular dynamics simulations support persistent gating loop - substrate interactions. The gating loop helps to position the substrate molecule to facilitate cellulose elongation (Verma et al. 2023).
Cellulose synthase complex of Arabidopsis thaliana
Cellulose synthase-like protein of 952 aas and about 7 TMSs, 3 (or 4) N-terminal and 6 C-terminal. May catalyze both beta-1,3 and beta-1,4 glycosidic linkages in beta-D-glucan. Essential for (1,3;1,4)-beta-D-glucans synthesis in grasses and cereals (Poaceae). The mixed-linked glucans (which are not present in walls of dicotyledons or most other monocotyledonous plants) are particularly important constituents of the walls of the starchy endosperm and aleurone cells of cereal grains such as oats, wheat, rice and barley. They can account for up to 70% by weight of the wall. A single amino acid within the predicted transmembrane pore domain of CslF6 controls (1-3,1-4)-beta-glucan structure. The membrane pore architecture and the translocation of the growing polysaccharide across the membrane control how the acceptor glucan is coordinated at the active site and thus the proportion of beta1-3 and beta1-4 bonds within the polysaccharide (Jobling 2015). Residues involved in catalyses and flexibility have been identified (Dimitroff et al. 2016). These influence the ratio of 1,3 to 1,4 linkages (Purushotham et al. 2022).
CslF6 of Oryza sativa subsp. japonica (Rice)
Cellulose synthase-like protein D2, CslD2, of 1145 aas and 8 TMSs in at 2 (N-terminal) plus 6 (C-terminal) arrangement.
CslD2 of Arabidopsis thaliana
Putative glycosyl transferase of 875 aas and 7 TMSs.
Fungi
UP of Sclerotinia sclerotiorum