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BESE Division Seminar - Dr. Maria Hrmova

Start Date: March 21, 2017
End Date: March 21, 2017

​​TITLE: Nanomolecular machines at work in plants: permeation and catalysis
DATE: Tuesday, March 21, 2017
TIME: 9:00 - 10:00 a.m.
LOCATION: Building 2 · Level 5 · Room 5220

Project #1: Transport proteins underlie plant adaptation or ‘taming’ transporters to grow hardier crops
Plants use transporters as vehicles to dispose of mineral substances, such as salt or boric acid, which are detrimental to plants at high concentrations [1]. In cereals, two types of membrane transporters have been implicated in toxicity tolerance caused by excess boric acid, which include root aquaporins of the nodulin-26-like intrinsic protein (NIP) type, and an anion-permeable transporter Bot1. Using an integrative platform of computational, biophysical, and biochemical tools, molecular biology, electrophysiology, and chemi- and bio-informatics, we investigate the origin of permeation function of both transporters [1-3].
The 3D model of the root anion-permeable transporter Bot1, supported by atomic force microscopy, revealed that it folds into 13 membrane-spanning and five cytoplasmic α-helices [1]. We predicted its trimeric assembly and the presence of a Na+ ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology and electrochemical impedance spectroscopy detected Na+-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using transport measurements, alanine scanning and molecular dynamics simulations, we showed that conductance of borate anions by Bot1 is abolished by the removal of the Na+ ion binding site [1].
The second class of proteins involved in boron toxicity tolerance include NIP aquaporins [2,3] that permeate water and boric acid in a neutral form. We described NIP’s permeation function and using molecular dynamics simulations we explained how in real time NIP conducts water, boric acid and remarkably sucrose.
Reference: [1] Nagarajan Y, Hrmova M, et al. (2016) Plant Cell 28:202. [2] Schnurbusch T Hrmova M, et al. (2010) Plant Phys 153:1706. [3] Luang S, Hrmova M (2017) Plant aquaporins, Springer-Verlag, ISBN 978-3-319-49393-0.
Project #2: The knowledge of enzyme molecular mechanisms underpins their use in biotechnology.
We investigate the molecular basis of catalysis in the glycoside hydrolase (GH) 3 family; these enzymes have enormous potential for applications in biotechnology. We use static and time-resolved crystallography, and other biochemical and biophysical techniques.
A broad specific GH3 β-D-glucan glucohydrolase is a two-domain protein consisting of an (α/β)8 barrel and an (α/β)6 sandwich (PDB accessions 1EX1, 1IEQ) [1,2] The active site of the enzyme is positioned in a pocket at the interface of two domains, and contains nucleophilic Asp285 and acid/base Glu491 residues, and an array of product/substrate binding residues (PDB 1X38, 1X39) [2]. The broad substrate specificity of this enzyme is rationalised from their crystal structures in complex with non-hydrolysable S-glycoside analogues (1IEX, 1J8V), prepared through organic and bio-organic chemistry [3,4]. However, the detailed binding mechanisms of (1,2)-, (1,3)-, (1,4)- and (1,6)-linked β-D-glucosides, that underlie the broad substrate specificity, are unknown. We assume that the flexibility of the subsite +1 between Trp286 and Trp434 may contribute to the accommodation of a variety of positional glucoside isomers.
A glucose moiety is observed in the active site of the β-D-glucan glucohydrolase and is the product of a hydrolytic reaction that serves as a natural inhibitor of catalysis. Structural mechanisms of product/substrate movements and the glucose inhibition remain poorly understood, and for this reason, this enzyme serves as a unique and convenient model for studying these processes. We suggest that protein-solvent interactions, and domain and side-chain residue movements, could play fundamental roles during product product/substrate movements. Information on the structural mechanisms of catalysis is applicable to various biotechnologies, such as biofuels production, paper and pulp, food, textile and detergent industries, and medical diagnostics.
References:[1] Hrmova M, et al. (2001) Structure 9:1005. [2] Hrmova M, et al. (2005) Biochemistry (USA) 44:16529. [3] Hrmova M, et al. (2002) Plant Cell 14:1033. [4] Luang S, Hrmova M, et al. (2010) Int J Mol Sci 11:2759.
Maria Hrmova is the Research Professor at the University of Adelaide, working in the field of plant structural biology. The goals of her projects are to elucidate in precise molecular terms, how key regulatory proteins (enzymes, transport proteins and transcription factors) operate during normal plant growth and in response to abiotic stresses. To tackle these challenges, she has developed an inter-disciplinary expertise in computational chemistry (3D protein molecular modelling), biophysics (X-ray crystallography and small-angle X-ray scattering) and biochemistry, and in molecular biology, electrophysiology, and chemi- and bio-informatics.
Since 2009 she leads the structural biology team at the University of Adelaide, focussing on the catalytic mechanisms of hydrolytic enzymes, and the mechanisms of transport proteins and transcription factors in plants.
Her key scientific achievements include the engineering of glycosynthetic enzymes, the dissection of catalytic mechanisms of β-D-glucanases, β-D-glucosidases, β-D-xylosidases and β-D-glucan exohydrolases involved in cereal grain germination, and landmark discoveries of hetero-trans-glycosylation in xyloglucan transferases underlying re-modelling of plant cell walls and cellulose synthase-like CslF enzymes playing roles in cell-wall biosynthesis. Further, she leads research focussing on the elucidation of the mode of action of HKT transporters involved in salt tolerance, and borate transporters and aquaporins underlying boron toxicity tolerance, and on the roles of transcription factors that regulate plant responses to water deficit. She also develops protein production methods for membrane proteins based on eukaryotic cell-free synthesis, and creates mimicking environments for membrane protein reconstitution.
She has co-authored 126 peer-reviewed articles and book chapters, lodged seven patents and published 22 articles in popular press. Advances of her research were highlighted on the front covers of 12 journals.
During her career at the University of Adelaide, she supervised 23 PhD, MSc and BSc completions, and secured funding for 19 Discovery, Linkage, Linkage-Infrastructure and Facility Equipment grants awarded by the Australian Research Council, and other funding bodies. The overall funding that she raised represents $12.1 million