Associate Professor Maria Hrmova
The University of Adelaide
Plant Genomics Centre
Waite Campus
Glen Osmond 5064

Personal Website Address: http://www.agwine.adelaide.edu.au/people/plant/mhrmo01.html
Email: maria.hrmova@adelaide.edu.au

Research Aims

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The Big Question

Determining the structure/function relationships of plant polysaccharide hydrolases and synthases using the techniques of crystallography and molecular modelling.

The Short-term goals

• Determining the 3D structures of the enzymes.
• Determining the 3D structures of the binary complexes of enzymes with their substrate analogues.
• Defining the catalytic and substrate-binding mechanisms.
• Defining the biological functions of the enzymes during normal plant development and in response to adverse effects.

Group(Lab Members and areas of research

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• Dr Hugues Driguez- (CERMAV, France) - carbohydrate chemistry and organic and bioorganic synthesis
• Dr Jose Varghese- (CSIRO, Melbourne) - crystallography and molecular modelling
• Professor Geoff Fincher - (ACPFG, Adelaide) - enzymology
• Dr Brian Smith - (WEHI, Melbourne) - Quantum mechanical calculations
• Dr Vladimir Farkas - (Institute of Chemistry, Slovak Republic) - enzymology
• Dr Peter Biely - (Institute of Chemistry, Slovak Republic) - enzymology.

Funding

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Current

ARC Discovery Grant " Molecular mechanisms of catalysis and the basis of substrate specificity in polysaccharide hydrolases".

Applications submitted

ARC Discovery Grant "Three-dimensional structures, substrate specificities and catalytic mechanisms of polysaccharide synthases".

Techniques in the Laboratory

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Currently active

• Protein purification and characterization
• Advanced enzyme kinetics including subsite mapping
• Protein sequencing
• Carbohydrate analysis
• In-gel enzyme activity identification
• Bioorganic synthesis of carbohydrates
• Glyco-synthetic technology
• Soluble protein crystallization
• Protein crystallography, molecular modelling and docking
• HPLC product and carbohydrate analyses
• Carbohydrate organic synthesis

In development

Fluorescent enzyme activity detection

• Crystallization of membrane-bound proteins
• Quantum mechanical calculations
• Time-resolved (Laue) crystallography

Top Three:

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Areas of knowledge

1. Enzyme mechanism and the basis of substrate specificity
2. Crystallography and 3D structures of catalytic proteins
3. Structure/function relationships of carbohydrate processing enzymes
4. Plant cell wall biosynthesis and degradation

Technical skills

1. Protein chemistry
2. Protein crystallography
3. 3D structures of catalytic proteins

A statement on most significant contributions to this research field

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The research during my scientific career has been focussing on plant and microbial enzymes, more specifically on carbohydrate-degrading enzymes. The projects addressed fundamental questions as to how polysaccharide hydrolases catalyse the hydrolysis of their substrates. In the past 10 years my work has been focussed mainly on barley endo- and exohydrolytic glycoside hydrolases, and during the last 6 years I have investigated ß- d -glucan glucohydrolases to define their catalytic mechanisms, thermodynamic and structural determinants of substrate binding. During the last 6 years we have identified, purified, cloned and crystallized a family 3 barley ß- d -glucan glucohydrolase. In collaboration with the CSIRO in Melbourne we have solved, formulated catalytic mechanism and defined the basis of substrate specificity for these exohydrolytic enzymes. During 2000-2003 we have analysed eight other three-dimensional (3D) structures in complexes with mechanism-based inhibitors and substrate analogues. These structures were subsequently used to provide structural and functional information on more than 180 members of the family 3 group glycoside hydrolases.

To achieve these goals I applied my expertise in protein/enzyme purification and characterization, in subsite mapping and other kinetic analyses, in molecular modelling, in chemoenzymic syntheses of oligosaccharides, and finally in the crystallization of proteins. More recently I have acquired knowledge of 3D structure solution by X-ray crystallography and solved several crystal structures of enzyme/ligand complexes. Further, computational methods such as molecular dynamics simulations and quantum mechanical calculations have been applied to various aspects of enzyme catalysis, and structural basis of substrate binding and specificity. Detailed knowledge of the catalytic mechanisms and of the molecular basis for substrate specificity is now applied to the generation of mutant enzymes (e.g. glycosynthases) with new catalytic activities and physicochemical properties such as enhanced stability, and to the specific syntheses of valuable oligosaccharides. We have recently prepared several mutant enzymes by site-directed mutagenesis. I believe that elucidating new enzyme structures and their interactions with mechanistic probes and substrates analogues has been an important contribution to the field, given that 3D structures are known for less than 20 plant enzymes. Finally, detailed 3D structural information will prove useful in the functional annotation of genes discovered in genome sequencing programs.

Other evidence of impact and contributions to the field

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Prizes: The presentation of the authors M. Hrmova, J.N. Varghese, R. DeGori, A.J. Harvey, H. Driguez and G.B. Fincher: "Crystal structures of barley ß -d -glucan exohydrolase comlexed with a substrate analogue and a mechanism-based inhibitor", was recognized at the Plant Polysaccharide Workshop in 2000 in Wageningen, Netherlands as "The most outstanding presentation".

Editorial board responsibilities: Significant part of my duties has involved the reviewing of manuscripts sent for publication in refereed journals including Nature Struct Biol, J Biol Chem, Biochim Biophys Acta , Austr J Plant Physiol , Plant Physiol , Planta , J Chem Soc , Perkin Trans , etc.

Experience in teaching and as committee member at the University of Adelaide, and BSc, MSc and PhD thesis assessor: Over the last 10 years, I have been involved in the training and supervision of 14 students at both undergraduate and postgraduate levels, and several postdoctoral fellows. I have also been a member of around 10 " ad hoc " intra- and inter-departmental committees associated with the judgement of job applicants within the Faculty of Sciences. Further, I have been appointed to assess BSc, MSc and PhD theses at the University of Adelaide and The University of Queensland.

Invited lectures and seminars and consultancy activities: I have been invited to present lectures to a broad scientific community, both at the University of Adelaide and other Australian universities, and internationally at the Centre de Recherches sur les Macromolecules Vegetales (Grenoble, France), University Joseph Fourier (Grenoble, France), Institute of Chemistry (Bratislava, Slovak Republic), Carlsberg Laboratories (Copenhagen, Denmark) and National Institutes of Health (Bethesda, USA). I have also been consulted by the Australian Wine Research Institute and CSIRO, Biochemistry Department at the University of Adelaide, Biotechnology Department at the Flinders University, etc. International institutions, with which I have collaborated include the University of Guelph (Guelph, Canada), University of Bern (Bern, Switzerland), Institute of Chemistry (Bratislava, Slovak Republic), University of Ramon Llul (Barcelona, Spain), National Institute of Agrobiological Resources (Tsukuba, Japan), Centre de Recherches sur les Macromolecules Vegetales (Grenoble, France), Carlsberg Laboratories (Copenhagen, Denmark), Manitoba University (Manitoba, Canada), and the Plant Biochemistry Laboratory at the Royal Veterinary and Agricultural University (Copenhagen, Denmark).

Chair of Australian and International Conferences and Symposia : 12th European Carbohydrate Symposium - EuroCarb 2003 (Session: Structure/function relationships of carbohydrate-degrading enzymes; Grenoble, France); ComBio 2004 Symposium Chair (Session: Enzyme mechanism; Perth, Australia).

Appointed: Working Group Meeting for the involvement of The University of Adelaide in National Synchrotron Program in Victoria.

Publications - Last 5 Years

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Books / Books Chapters

• Hrmova M , Fincher GB (2003) Enzymic hydrolysis of cereal (1,3;1,4)-ß-glucans. In: Handbook of Food Enzymology , (Whitaker JR, Voragen AGJ, Wong DWS, eds), Marcel Dekker, New York, 2003, pp 943-960. Invited review.

• Hrmova M , Fincher GB (2002) Three-dimensional structures, substrate specificities and biological functions of ß -d- glucan endo- and exohydrolases from higher plants. In: Plant Cell Walls , (Carpita NC, Cambell M, Tierney M, eds), Wolter Kluwer Academic Publishers, Dordrecht, Netherlands, 2002, pp 73-91. Invited review.

Journal Articles

• Zhang D, Balzen J, Wu C, Wan CH, Cai W, Wang J, Zhang H, Hrmova M , Fincher GB, Haigler CH (2004) Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls. Plant Molecular Biology, 2004, in the press (accepted Feb 2004).

• Macdonald JM, Hrmova M , Fincher, GB, Stick RV (2004) The synthesis of 3- O -(ß- d -glucopyranosyl)- and 3- O -(ß-laminaribiosyl)-isofagomine: Putative inhibitors of 1,3-ß- d -glucan endohydrolases. Australian Journal of Chemistry, 57,187-191.

• Hrmova M , De Gori R, Smith BJ, Vasella A, Varghese JN, Fincher GB (2004) Three-dimensional structure of the barley ß- d -glucan glucohydrolase in complex with a transition-state mimic. Journal of Biological Chemistry, 279, 4970-4980.

• Li J, Burton RA, Harvey AJ, Hrmova M , Wardak AZ, Stone BA, Fincher GB (2003) Biochemical evidence linking a putative callose synthase gene with (1,3)-ß- d- glucan biosynthesis in barley. Plant Molecular Biology, 53, 213-225.

• Hrmova M , Imai T, Rutten SJ, Fairweather JK, Pelosi L, Bulone V, Driguez H, Fincher GB (2003) Barley (1,3)-ß -d- glucan endohydrolase mutants synthesise crystalline (1,3)-ß -d- glucans. Journal of Biological Chemistry 278, 48506.

• Fairweather J, Hrmova M , Rutten, SJ, Fincher GB, Driguez D (2003) Synthesis of complex oligosaccharides using a mutated (1,3)-ß -d -glucan endohydrolase from barley. Chemistry - A European Journal 9, 2603-2610.

• Lee RC, Hrmova M , Burton RA, Lahnstein J, Fincher GB (2003) An a- l -arabinofuranosidase and a ß- d -xylosidase from barley: Purification, characterization and primary structures. Journal of Biological Chemistry 278, 5377-5387.

• Hrmova M , Imai T, Rutten SJ, Fairweather JK, Pelosi L, Bulone V, Driguez H, Fincher GB (2002) Barley (1,3)-ß -d- glucan endohydrolase mutants synthesise crystalline (1,3)-ß -d- glucans. Journal of Biological Chemistry 277, 30102-30111.

• Hrmova M , De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, Fincher GB (2002) Structural basis for a broad specificity in higher plant ß -d -glucan glucohydrolases. The Plant Cell 14,1033-1052. Feature article with a front cover photograph.

• Hrmova M , Varghese JN, DeGori R, Smith BJ, Driguez H, Fincher GB (2001) Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of plant ß -d -glucan glucohydrolase. Structure 9 , 1015-1016.

• Hrmova M , Fincher, GB (2001) Structure-function relationships of ß -d -glucan endo- and exohydrolases from higher plants. Plant Molecular Biology 47, 73-91. Invited review.

• Osmond RIW, Hrmova M , Fontaine F, Imberty A, Fincher GB (2001) Barley thaumatin-like proteins: ligand specificity, kinetic and structural analyses and biological implications. European Journal of Biochemistry 268, 4190-4199.

• Harvey AJ, Hrmova M , Fincher GB (2001) Regulation of genes encoding ß -d -glucan endohydrolases in barley ( Hordeum vulgare ). Physiologia Plantarum 113, 108-120.

• Hrmova M, Fincher GB (2001) Plant enzyme structure: explaining substrate specificity and the evolution of function. Plant Physiology 125, 54-57. Invited review.

• Lee RC, Burton RA, Hrmova M, Fincher GB (2001) Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones. Biochemical Journal 356, 181-189.

• Harvey AJa, Hrmova Ma, DeGori R, Varghese JN, Fincher GB (2000) Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases. Proteins: Structure, Function & Gene tics 41, 257-269. aContributed as the equal authors.

• Hrmova M, Stewart RJ, Varghese JN, Høj PB, Fincher GB (1999) Three-dimensional structures, catalytic mechanisms and protein engineering of ß -d -glucan exohydrolases from barley. Recent Advances in Carbohydrate Bioengineering 246, 124-131.

• Varghese JN, Hrmova M, Fincher GB (1999) Three-dimensional structure of a barley ß -d -glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 7, 179-190.

• Burton RA, Zhang XQ, Hrmova M, Fincher, GB (1999) A single limit dextrinase gene is expressed both in the developing endosperm and in germinated grains of barley. Plant Physiology 119 , 859-871.

Ten career-best publications

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• Hrmova M , De Gori R, Smith BJ, Fairweather JK, Driguez H, Varghese JN, Fincher GB (2002) Structural basis for a broad specificity in higher plant ß -d -glucan glucohydrolases. The Plant Cell 14, 1033-1052. Feature article with a front cover photograph.

• Hrmova M , Varghese JN, DeGori R, Smith BJ, Driguez H, Fincher GB (2001) Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of plant ß -d -glucan glucohydrolase. Structure 9, 1015-1016.

• Varghese JN, Hrmova M , Fincher GB (1999) Three-dimensional structure of a barley ß -d -glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 7, 179-190.

• Hrmova M , Fincher, GB (2001) Structure-function relationships of ß -d -glucan endo- and exohydrolases from higher plants. Plant Molecular Biology 47, 73-91. Invited review.

• Hrmova M , Fincher GB (2003) Enzymic hydrolysis of cereal (1,3;1,4)-ß-glucans. In: Handbook of Food Enzymology , (Whitaker JR, Voragen AGJ, Wong DWS, eds), Marcel Dekker, New York, pp. 943-960. Invited review.

• Hrmova M , De Gori R, Smith BJ, Vasella A, Varghese JN, Fincher GB (2004) Three-dimensional structure of the barley ß- d -glucan glucohydrolase in complex with a transition-state mimic. Journal of Biological Chemistry , 279, 4970-4980.

• Hrmova M , Imai T, Rutten SJ, Fairweather JK, Pelosi L, Bulone V, Driguez H, Fincher GB (2002) Barley (1,3)-ß -d- glucan endohydrolase mutants synthesise crystalline (1,3)-ß -d- glucans. Journal of Biological Chemistry 277, 30102-30111.

• Hrmova M , MacGregor EA, Biely P, Stewart RS, Fincher GB (1998) Substrate binding and catalytic mechanism of a barley ß -d -glucosidase/(1,4)-ß -d -glucan exohydrolase. Journal of Biological Chemistry 273, 11134-11143.

• Hrmova M , Harvey AJ, Wang J, Shirley NJ, Jones GP, Høj PB, Fincher GB (1996) Barley ß -d -glucan exohydrolases with ß -d -glucosidase activity. Purification and determination of primary structure from a cDNA clone. Journal of Biological Chemistry 271, 5277-5286.

• Hrmova M , Garrett TPJ, Fincher GB (1995) Subsite affinities and disposition of catalytic amino acids in the substrate-binding region of barley 1,3-ß -d -glucanases. Journal of Biological Chemistry 270, 14556-14563.