Engineering Papers⌕ Search

Engineering topics

Ho, Ruoya

Publications and source records attributed to Ho, Ruoya.

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Insights into substrate coordination and glycosyl transfer of poplar cellulose synthase-8

Cellulose is an abundant cell wall component of land plants. It is synthesized from UDP-activated glucose molecules by cellulose synthase, a membrane-integrated processive glycosyltransferase. Cellulose synthase couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Here, we present substrate- and product-bound cryogenic electron microscopy 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 reveals its critical function for catalytic activity. Molecular dynamics simulations reveal 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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Mechanism of mixed-linkage glucan biosynthesis by barley cellulose synthase–like CslF6 (1,3;1,4)-β-glucan synthase

Mixed-linkage (1,3;1,4)-β-glucans, which are widely distributed in cell walls of the grasses, are linear glucose polymers containing predominantly (1,4)-β-linked glucosyl units interspersed with single (1,3)-β-linked glucosyl units. Their distribution in cereal grains and unique structures are important determinants of dietary fibers that are beneficial to human health. We demonstrate that the barley cellulose synthase-like CslF6 enzyme is sufficient to synthesize a high–molecular weight (1,3;1,4)-β-glucan in vitro. Biochemical and cryo–electron microscopy analyses suggest that CslF6 functions as a monomer. A conserved “switch motif” at the entrance of the enzyme’s transmembrane channel is critical to generate (1,3)-linkages. There, a single-point mutation markedly reduces (1,3)-linkage formation, resulting in the synthesis of cellulosic polysaccharides. Our results suggest that CslF6 monitors the orientation of the nascent polysaccharide’s second or third glucosyl unit. Register-dependent interactions with these glucosyl residues reposition the polymer’s terminal glucosyl unit to form either a (1,3)- or (1,4)-β-linkage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales)

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. Furthermore, the relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Molecular organization of the E. coli cellulose synthase macrocomplex

Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA–B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA’s periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA’s regulatory PilZ domain. As a result, the macrocomplex is a fascinating example of cellulose synthase specification.

59 BASIC BIOLOGICAL SCIENCES↗