Unravelling Regioselectivity of Leuconostoc citreum ABK-1 Alternansucrase by Acceptor Site Engineering

Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose–acceptor reaction—as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.

Effect of exogenously-applied abscisic acid, putrescine and hydrogen peroxide on drought tolerance of barley

The objective of this study was to identify the effect of abscisic acid (ABA), putrescine (Put) and hydrogen peroxide (H2O2) foliar pre-treatment on drought tolerance of barley. Despite water limitation, ABA-sprayed plants preserved increased water content, photosynthetic efficiency of PSII (ΦPSII) and CO2 assimilation rate (Pn) compared to untreated stressed plants. The ABA-treated plants presented also the lowest rate of lipid peroxidation (MDA), lowered the rate of PSII primary acceptor reduction (1 – qP) and increased the yield of regulated energy dissipation (NPQ) with higher accumulation of PGRL1 (PROTON GRADIENT REGULATION LIKE1) protein. These plants preserved a similar level of photochemical efficiency and the rate of electron transport of PSII (ETRII) to the well-watered samples. The significantly less pronounced response was observed in Put-sprayed samples under drought. Additionally, the combined effects of drought and H2O2 application increased the 1 – qP and quantum yield of non-regulated energy dissipation in PSII (ΦNO) and reduced the accumulation of Rubisco activase (RCA). In conclusion, ABA foliar application allowed to balance water retention and preserve antioxidant capacity resulting in efficient photosynthesis and the restricted risk of oxidative damage under drought. Neither hydrogen peroxide nor putrescine has been able to ameliorate drought stress as effectively as ABA.

Ovule development in Cycads: observation on anatomy and nucellus morphology in Zamia and Cycas

In the ovule evolution, the integument is the most attention point in discussion as a morphologic character of the seed plants. There are several theories and hypotheses about the origin of the integument were presented in the history. However, the development and function of the ovule envelopes are not so clear until now. The development of thehe basal gymnosperms Cycas and Zamia were to investigated, especially of the integument to complement the existing knowledge in seed plants. The development of ovules of seed plant is documented with morphological and anatomical using LM and SEM.The nucellar beak found in Zamia is a structure that has not been recorded previously. It protrudes from the micropyle at pollination and may be the primary acceptor for pollen. There are striking similarities to the lagenostom or salpinx in Lyginopteridatae. There may be an evolutionary way to interpret the pollination drop existing in the Lyginopteridatae. Probably the nucellar beak of Cycads, even Ginkgoales have the same function with the lagenostom or salpinx of the Lyginopteridatea. Unfortunately, pollen and transport inside the pollination chambers have not been observed. Further analysis of this unusual structure seems to be very important.

Towards a full understanding of water splitting in photosynthesis

The capture and conversion of solar radiation by photosynthetic organisms directly or indirectly provides energy for almost all life on our planet. About 2.5 billion years ago a remarkable biological “machine” evolved known as photosystem two (PSII). This machine can use the energy of visible light (actually red quanta of∼1.8 eV) to split water into dioxygen and “hydrogen”. The latter is made available as reducing equivalents, ultimately destined to convert carbon dioxide to organic molecules. In PSII, the “hydrogen” reduces plastoquinone (PQ) to plastoquinol(PQH2). The water splitting process takes place at a catalytic centre composed of 4 Mn atoms and the reactions involved are chemically and thermodynamically challenging. The process is driven by a photooxidised chlorophyll molecule(P680•+)and involves electron/proton transfer reactions aided by a redox active tyrosine residue situated between the 4 Mn cluster and P680. TheP680•+species is generated by light induced rapid electron transfer (a few picoseconds) to a primary acceptor, pheophytina, before being transferred to PQ acceptors. Electron and x-ray crystallographic studies are now starting to reveal the structural basis for these reactions including the light harvesting processes. The 4 Mn atom-cluster has been visualised as have the chlorophylls that constitute P680. The scene is now set to fully elucidate the reactions of PSII and possibly mimic them in an artificial photochemical system that could split water and produce hydrogen.