Understanding the mechanism of force generation in the bacterial flagellar motor
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, small angle X-ray scattering and FRET. The ongoing research aims to unravel the mechanism of stator assembly, anchoring to the peptidoglycan and force generation.
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.
How does H. pylori sense environmental cues?
H. pylori chemotaxis, mediated by chemoreceptors, plays an important role in initial colonization and development of disease. We investigate what ligands such receptors recognize and how, why some molecules are attractants and some - repellents, how binding to the receptor leads to signalling, how mutations in the sensor domain affect ligand specificity and, building on this, how bacterial chemoreceptors can be redesigned to recognise and respond to non-native ligands for innovative applications in biotechnology and bioengineering.
New targets for the old drugs: exploring the antimicrobial potential of carbonic anhydrase inhibitors
H. pylori has a unique ability to withstand high acidity of the stomach by buffering its periplasm at pH 6, through the action of urease and carbonic anhydrase (CA). We evaluate the potential of H. pylori CA as a novel target for treatment against Helicobacter. We have determined the first crystal structure of this enzyme in complex with inhibitors that have been used clinically for a different purpose, i.e. as antiglaucoma or antiulcer drugs. The study includes analysis of structure-activity relationships, isolation of mutants with spontaneous resistance and genomic investigation of the resistance mechanisms.
Prospective PhD students
PhD scholarships are available to Australian and international applicants; specific information on how to apply to the Monash postgraduate programme, eligibility and application deadlines can be found here.
Selected Recent Publications
• Ud-Din AI, Tikhomirova A and Roujeinikova, A (2016) Structure and functional diversity of GCN5-related N-acetyltransferases (GNAT). Int. J. Mol. Sci. 17, 1018
• Machuca MA, Liu YC, Beckham SA, Gunzburg MJ and Roujeinikova A (2016) The crystal structure of the tandem-PAS sensing domain of Campylobacter jejuni chemoreceptor Tlp1 suggests indirect mechanism of ligand recognition. J. Struct. Biol. 194, 205-213.
• Modak JK, Rut W, Wijeyewickrema LC, Pike RN, Drag M and Roujeinikova A (2016) Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase. Biochimie 121, 60-71.
• Liu YC, Machuca MA, Beckham SA, Gunzburg MJ and Roujeinikova A. (2015) Structural basis for amino-acid recognition and transmembrane signalling by tandem Per-Arnt-Sim (tandem PAS) chemoreceptor sensory domains. Acta Cryst. D71, 2127-2136.
• Modak JK, Liu YC, Machuca MA, Supuran CT and Roujeinikova A. (2015) Structural basis of inhibition of Helicobacter pylori α-carbonic anhydrase by sulfonamides. PLoS ONE 10, e0127149.
• Ud-Din AI, Liu YC and Roujeinikova A. (2015) Crystal structure of Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH: implications for substrate specificity and catalysis. PLoS ONE 10, e0115634.
• Alonso H, Kleifeld O, Yeheskel A, Ong PC, Liu YC, Stok JE, De Voss JJ, Roujeinikova A. (2014) Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB. Biochem. J. doi:10.1042/BJ20131648.
• Narayanan S, Modak JK, Ryan CS, Garcia-Bustos J, Davies JK, Roujeinikova A. (2014) Mechanism of Escherichia coli resistance to pyrrhocoricin. Antimicrob Agents Chemother. 58, 2754-2762.
• Andrews DA, Xie M, Hughes V, Wilce MC & Roujeinikova A (2013) Design, purification and characterisation of a soluble variant of the integral membrane protein MotB for structural studies. J. Royal Soc. Int. 10, 20120717.
• Woon AP, Tohidpour A, Alonso A, Saijo-Hamano, Y., Kwok T. and Roujeinikova A. (2013) Conformational analysis of isolated domains of Helicobacter pylori CagA. PLoS ONE 8, e79367.
• Alonso H & Roujeinikova A (2012) Characterisation and 2D crystallisation of the membrane component AlkB of the medium-chain alkane hydroxylase system from Pseudomonas putida GPo1. Appl. Environ. Microbiol. 78, 7946-7953.
• O’Neill J, Xie M, Hijnen M & 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 & 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.
• Reboul CF, Andrews DA, Nahar MF, Buckle AM & 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.
A/Prof Roujeinikova’s full list of publications
Roujeinikova Group Members
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Assoc Prof Anna Roujeinikova |
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Joyanta Modak (PhD Student) |
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Mayra Machuca-Perez (PhD Student) |
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Mizanur Rahman (PhD Student) |
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Alexandra Tikhomirova (Research Fellow) |
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Abu Salah Ud-Din (PhD Student) |
