Anna Roujeinikova's laboratory

Anna Roujeinikova Associate Professor ARC Research Fellow Phone: +61 3 9902 9194 Fax: +61 3 9902 9222 E-mail: Anna.Roujeinikova@monash.edu Postal address: Building 76, School of Biomedical Sciences, Monash University, Clayton, VIC 3800, Australia

BSc, Natural Sciences (1994, Moscow Institute of Physics and Technology, Russia)
MSc, Applied Physics and Mathematics (1997, Moscow Institute of Physics and Technology)
PhD, Structural Biology (2002, University of Sheffield, UK)
Wellcome Trust Research Career Development Fellowship (2006-2009, University of Manchester, UK)

Keywords: Helicobacter pylori, bacterial flagellar motor, virulence factors, X-ray crystallography, protein structure, molecular machines, protein-protein and protein-substrate recognition, alkane hydroxylases, biodegradation.

Our main research focus is structural biology of virulence factors of the carcinogenic bacterium Helicobacter pylori. H. pylori must be able to swim by means of its flagella in order to infect the human host and persist for years in the gastric mucosa. We study the mechanism of force generation in H. pylori flagellar motor. Development of gastric cancer in infected individuals is facilitated by exposure of gastric cells to H. pylori protein CagA. We investigate the mechanism of CagA-mediated gastric cell transformation.

Understanding the function of the motility protein B component of the bacterial flagellar motor (BFM)

The bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim toward nutrients, optimal temperatures, or other factors that favour survival. Motility by flagellar motor is essential for the survival, chemotaxis and virulence of many pathogenic bacteria. The chosen model system, the carcinogenic bacterium Helicobacter pylori, uses the flagellar motor to drill into the mucus layer of the stomach and move towards the epithelial surface, where it colonizes. The molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the individual components of the motor's stator unit. The highly dynamic nature of the stator complex creates the biggest challenge in obtaining its structural information. We are taking both top-down and bottom-up approaches to this problem, combining data from the molecular genetics studies, cross-linking, X-ray protein crystallography, electron microscopy, peptide amide hydrogen/deuterium exchange coupled with liquid chromatography and mass spectrometry. We have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the bacterial flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. The ongoing research aims to unravel the mechanism of stator assembly, anchoring to the peptidoglycan and force generation.

Electron density for N-acetylmuramic acid bound to the MotB peptidoglycan-binding domain and the model for the glycan chain binding.

The role and the mechanism of bacterial born carcinogen CagA

Pathogenic strains of H. pylori, associated with the development of adenocarcinoma in humans, inject CagA protein into gastric epithelial cells, where it interacts with many different host cell proteins, interfering with signalling pathways that regulate cell growth and motility. We undertake detailed characterization of the CagA structure and interactions for elucidation of its rolein gastric carcinogenesis. In complementing experiments, we assess the in-vivo activity of CagA fragments (e.g. effect on cell morphology and motility, interaction with various partner molecules).

Our second research interest is the mechanism of the biotechnologically important enzyme alkane hydroxylase.

Understanding the structural basis for catalysis and substrate specificity in non-heme diiron medium-chain alkane hydroxylases

Bacterial alkane hydroxylases are of high interest for bioremediation studies as they allow some bacteria to grow on oily wastes and in oil-contaminated, alkane-rich environments. They also have tremendous biocatalytic potential as tools for specific transformation of alkanes into the building blocks that can be used for synthesis of pharmaceuticals and other high cost chemicals. We aim to determine the molecular basis for catalysis and specificity of nonheme diiron medium-chain alkane hydroxylase by solving its first crystal structure and by carrying out complementary biochemical experiments with chemical probes. Identification of the active site residues will pave the way to rational design of this enzyme with the aim to increase its biocatalytic potential for modification of industrially important chemical precursors and biodegradation of spilt oils.

An electron micrograph and reconstruction map of negatively stained 2D crystals of membrane-reconstituted alkane hydroxylase

Joining the lab:

2012 Lab members:

• Hernan Alonso (postdoctoral fellow)
• Daniel Andrews (PhD student)
• Abolghasem Tohidpour (PhD student)
• Shalini Narayanan (PhD student)
• Amanda Woon (Honours student)

Prospective PhD students:

A range of other Ph.D. scholarships are open to Australian and international students; specific information on how to apply to the Monash postgraduate programme, eligibility and application deadlines can be found here. International students can also apply through the CCV, Endeavour, CSIRO.

Prospective postdoctoral fellows:

Australian postdoctoral researchers and PhD students in their final year are encouraged to apply through the available fellowship schemes (Discovery Early Career Researcher Award (DECRA), NHMRC training (postdoctoral) fellowship). International candidates should consult HFSP and EMBO web pages for open fellowship schemes.

Recent publications

O’Neill, J., Xie, M., Hijnen, M. and Roujeinikova, A. (2011) Role of the MotB linker in the assembly and activation of the bacterial flagellar motor. Acta Cryst. D 67, 1009-1016.

Aydin, I., Saijo-Hamano, Y., Namba, K., Thomas, C. and Roujeinikova, A. (2011) Structural analysis of the essential resuscitation promoting factor YeaZ suggests a mechanism of nucleotide regulation through dimer reorganization. PLoS ONE 6, e23245.

Xie, M., Alonso, H. and Roujeinikova, A. (2011) An improved procedure for the purification of catalytically active alkane hydroxylase from Pseudomonas putida GPo1. Appl. Biochem. Biotechnol. 165, 823-831.

Reboul, C.F., Andrews, D.A., Nahar, M.F., Buckle, A.M. and Roujeinikova, A. (2011) Crystallographic and molecular dynamics analysis of loop motions unmasking the peptidoglycan-binding site in stator protein MotB of flagellar motor. PLoS ONE 6, e18981.

Aydin, I., Dimitropoulos, A., Chen, S.H., Thomas, C. and Roujeinikova, A. (2011) Purification, crystallization and preliminary X-ray crystallographic analysis of the putative Vibrio parahaemolyticus resuscitation promoting factor YeaZ. Acta Cryst. F 67, 604-607.

Liebscher, M. and Roujeinikova, A. (2009) Allosteric coupling between the lid and the interdomain linker in DnaK revealed by inhibitor binding studies. J. Bacteriol. 191, 1456-1462.

Toogood, H.S., Hare, V., Fryszkowska, A., Fisher, K., Roujeinikova, A., Heyes, D.J., Leys, D., Stephens, G.M., Gardiner, J. and Scrutton, N.S. (2008) Structure-based insight into the asymmetric bioreduction of the C═C double bond of α,β-unsaturated nitroalkenes by pentaerythritol tetranitrate reductase. Adv. Synth. Catal. 350, 2789-2803.

Hothi, P., Hay, S., Roujeinikova, A., Sutcliffe, M.J., Lee, M.., Cullis, P.M. and Scrutton, N.S. (2008) Driving force analysis of proton tunnelling across a reactivity series for an enzyme-substrate complex. ChemBioChem. 9, 2839-2845.

Roujeinikova, A. (2008) Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB. Acta Cryst. F 64, 277-280.

Hothi, P., Roujeinikova, A., Sutcliffe, M.J., Cullis, P., Leys, D. and Scrutton, N.S. (2007) Isotope effects reveal that p-substituted benzylamines are poor reactivity probes of quinoprotein mechanism for aromatic amine dehydrogenase. Biochemistry 46, 9250-9259.

Roujeinikova, A., Hothi, P., Scrutton, N.S. and Leys, D. (2007) New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates. J. Biol. Chem. 282, 23766-23777.