• peptidoglycan deacetylases
• chitin deacetylases
Polysaccharide deacetylases belong to Carbohydrate Esterase Family 4 (CE4) which includes chitin deacetylases, acetyl-xylan esterases, xylanases, rhizobial NodB chitooligosaccharide deacetylases and peptidoglycan deacetylases. All these enzymes share a universal conserved region called polysaccharide deacetylase domain (according to the Henrissat classification). All five members of this family catalyze the hydrolysis of either N -linked acetyl group from N -acetylglucosamine residues (chitin deacetylases, NodB and peptidoglycan N -acetylglucosamine deacetylases), or O -linked acetyl groups from O -acetylxylose residues (acetyl xylan esterases, xylanases).
Peptidoglycan modification, specifically N -deacetylation, is a highly efficient strategy used by pathogenic bacteria to evade innate host defenses. For example, de- N -acetylation of peptidoglycan GlcNAc confers resistance to lysozyme, an exogenous muramidase, upon several bacterial species, such as S. pneumoniae, Bacillus cereus, L. monocytogenes, Lactococcus lactis and Helicobacter pylori.
The recent sequencing of the B. cereus and B. anthracis genomes revealed in each a multiplicity of putative polysaccharide deacetylases. Six of these genes have been proposed to encode for putative peptidoglycan N -acetylglucosamine deacetylases in B. cereus and have almost identical amino acid sequence with the corresponding ones from B. anthracis implying similar functional roles of these proteins in the two bacteria.
Peptidoglycan N-deacetylation occurs at N-linked acetyl groups of GlcNAc or MurNAc(oloured in red)
We employ a combined biochemical, crystallographic and genetic (knock out) analysis for these studies. Protein crystallization experiments are performed in collaboration with Prof D. Christianson group. The objective of this study is to shed light on the role of bacterial peptidoglycan deacetylases and furthermore based on the extensive homologies to contribute to our understanding of the physiology of B. anthracis, an interesting pathogenic microbe close relative to B. cereus.
The long term goal is to develop drugs that inhibit the enzymes. This should make important human pathogenic bacteria such as B. anthracis susceptible to lysozyme in the blood and provide an alternative therapeutic strategy.
1. Purification, crystallization and preliminary X-ray analysis of the peptidoglycan N-acetylglucosamine deacetylase BC1960 from Bacillus cereus in the presence of its substrate (GlcNAc) 6 ” A. TSALAFOUTA, E. PSYLINAKIS, E.G. KAPETANIOU, D. KOTSIFAKI, A. DELI, A. ROIDIS, V. BOURIOTIS and M. KOKKINIDIS, (2008), Acta Crystallographica Section F: F64, 203–205
2. A critical role for peptidoglycan N -deacetylation in Listeria evasion from the host innate immune system” I. G. BONECA, O. DUSSURGET, D. CABANESC, M-A. NAHORIC, S. SOUSA, M. LECUIT, E. PSYLINAKIS, V. BOURIOTIS, J-P. HUGOTI, M. GIOVANNINIK, A. COYLEM, J. BERTINM, A. NAMANEP, J-C. ROUSSELLE, N. CAYET, M-C. PREVOST, V. BALLOY, M. CHIGNARD, D. J. PHILPOTT, P. COSSART, and S. E. GIRARDIN (2007), PNAS vol.104, no.3, 997–1002
3. Peptidoglycan N- Acetylglucosamine Deacetylases from Bacillus cereus, Highly Conserved Proteins in Bacillus anthracis” E. PSYLINAKIS, I.G. BONECA, K. MAVROMATIS, A. DELI, E. HAYHURST, S. J. FOSTER, K. M. VARUM and V. BOURIOTIS (2005) : J.Biol.Chemistry (280),(35) , 30856-30863
Our group has a long tradition in the study of chitin deacetylases from fungi (Colletotrichum lindemuthianum, Mucor rouxii, Saccharomyces cerevisiae). We have recently focused on the study of chitin deacetylases from Cryptococcus neoformans. In the framework of an EU project a major effort was undertaken in the characterization of these enzymes.
Chitin is deacetylated by the enzyme chitin deacetylase to form chitosan and acetate.
Cryptococcus neoformans is an encapsulated yeast that can infect both immunocompetent individuals and patients with a number of defects in antimicrobial defenses. Cryptococcosis has emerged as one of the most frequent life-threatening infections in AIDS patients.
Conventional antifungal therapy has major limitations and patients may progress to fatal meningoencephalitis despite chronic treatment. Therefore there is a need to develop alternative strategies to control Cryptococcosis. This enzyme has been shown to be able to produce not only delayed hypersensitivity reactions but also protective immunity responses. The gene (d25) encoding for one of the four putative chitin deacetylases has been expressed in K. lactis and the recombinant enzyme purified to homogeneity and further characterized.
Further studies are needed to test the hypothesis that the protein described is involved in cell wall formation and is therefore a potential target for chemotherapy. D25 crystal structure studies are in progress by Dr P. Heikinheimo.
1. Expression, characterization and functional analysis of a recombinant chitin deacetylase secreted in the periplasmic space of E. coli” A. MARTINOU, D. KOUTSIOULIS, and V. BOURIOTIS, (2003), Enzyme and Microbial Technology: 32, 757-763
2. Chitin deacetylases:new, versatile tools in biotechnology” I. TSIGOS, A. MARTINOU, D. KAFETZOPOULOS and V. BOURIOTIS, (2000), Trends in Biotechnology: 18, 7, 305-312
3. Mode of action of chitin deacetylase from Mucor rouxii on N acetylchitooligo-saccharides” I.TSIGOS, N. ZYDOWICZ, A. MARTINOU, A. DOMARD V. BOURIOTIS (1999), Eur. J. Biochem.: 261, 698-705
LmbE family proteins
Only a few members of the LmbE protein family have been biochemically characterized including N -acetylglucosaminylphosphatidyl inositol (GlcNAc-PI), 1-D-myo-inosityl-2-acetamido-2-deoxy- a -D-glucopyranoside (GlcNAc-Ins), N',N' -diacetylchitobiose (GlcNAc 2 ) and lipoglycopeptide antibiotic de- N -acetylases.
LmbE proteins substrates. All enzymes catalyze hydrolysis of the N-acetyl group (shown inside a circle or a rectangle) of the GlcNAc moiety of their substrate(s).
The enzymes presently identified and characterized are involved in biosynthesis of glycophosphatidyloinositol, mycothiol and a novel chitinolytic pathway in archaea respectively and have tremendous potential for biotechnological applications either in the design of antimicrobial inhibitors or the production of novel chitooligomers and in particular glucosamine. All these enzymes share a common feature in that they de- N -acetylate the N -acetyl-D-glucosamine (GlcNAc) moiety of their substrates.
The genomes of B. cereus and its closest relative B. anthracis each contain two LmbE protein family homologs: BC1534 (BA1557) and BC3461 (BA3524). The objective of this study is to shed light on the role of two de- N -acetylases from B. cereus, which are LmbE protein family homologs. Given the extensive homology between B. cereus and B. anthracis, these studies could contribute to our understanding of the physiology of this interesting pathogenic microbe. The crystal structure of BcZBP protein from B. cereus has been determined by Prof. M. Kokkinidis group.
Structure of BcZBP protein from B. cereus.
1. LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and highly conserved proteins in Bacillus anthracis” A. DELI, D. KOUTSIOULIS, P. SPILIOTOPOULOU, K. MAVROMATIS, V.FADOULOGLOU, M. KOKKINIDIS and V. BOURIOTIS (2010), FEBS Journal: (277), 2740-2753.
2. Molecular Dynamics Simulations of BcZBP, A Deacetylase from Bacillus cereus: Active Site Loops Determine Substrate Accessibility and Specificity” V.E. FADOULOGLOU, A. STAVRAKOUDIS, V. BOURIOTIS, M. KOKKINIDIS and N. GLYKOS, (2009), J. Chem. Theory Comput., 2009, 5 (12), pp 3299-3311
3. Crystal structure of the BcZBP, a zinc-binding protein from Bacillus cereus” V.E. Fadouloglou, A. DELI, N.M, GLYKOS, E. PSYLINAKIS, V. BOURIOTIS and M. KOKKINIDIS (2007), Functional insights from structural data (FEBS Journal): 274, 3044-3054
Cold adapted enzymes
Psychrophiles or cold-loving organisms successfully colonize cold environments of the Earth's biosphere. To cope with the reduction of chemical reaction rates induced at low temperatures, these organisms synthesize enzymes characterizised by a high catalytic activity at low temperatures associated, however, with low thermal stability. Due to their attractive properties these enzymes have tremendous potential for fundamental research and biotechnological applications. Our group has a long tradition in the study of cold adapted enzymes.
We have focused on the study of three cold adapted enzymes namely an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp TAE123, a chitnase from the Antarctic Arthrobacter sp. TAD20 and an alkaline phosphatase from the Antarctic strain TAB5.
The NAD+- dependent psychrophilic alcohol dehydrogenase (ADH) has been produced in E. coli, purified to homogeneity and further characterized.
In collaboration with Prof. Klimman's group it was shown that the enzyme exhibits distinctive catalytic parameters in relation to the homologous thermophilic ADH from B. stearothermophilus. The crystal structure of the native ADH as well as its complex with NADH have been determined by Dr. Petratos group and refined to 2.2Å resolution.
Employing site directed mutagenesis, mutations were designed in an attempt to introduce a salt bridge and replace selected Gly residues by Pro respectively in a cold adapted chitinase. The mutants obtained exhibited higher thermal stability as compared to the wild type protein
In an effort to engineer cold adapted biocatalysts to operate at elevated temperatures we have employed both rational redesign and directed evolution on the psychrophilic alkaline phosphatase from the Antarctic strain TAB5.
3D- structure of alkaline phosphatase from the Antarctic strain TAB5
In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase. The results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild type sequence, which may affect active site flexibility through changes in various conformational constrains.
Furthermore, in an effort to explore the role of glycine clusters on the cold adaptation of enzymes we designed point mutations aiming to alter the distribution of glycine residues close to the active site of the psychrophilic alkaline phosphatase. It appears that the Gly cluster in combination with its structural environment plays a significant role in the cold adaptation of the enzyme. The crystal structure of alkaline phosphatase has been determined and refined to 1.8Å resolution by Prof. P. Heikinheimo group.
Finally using directed evolution several alkaline phosphatase mutants were obtained exhibiting either higher activity as compared to the wild type protein or higher thermal stability.
Schematic presentation of directed evolution studies
1. Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase” D. KOUTSIOULIS, E. WANG, M. TZANODASKALAKI, D. NIKIFORAKI, A. DELI, G. FELLER, P. HEIKINHEIMO, and V. BOURIOTIS (2008), Protein Engineering, Design&Selection: vol.21 no.5 pp. 319-327
2. Crystal Structure of Alkaline Phosphatase from the Antarctic Bacterium TAB5” E. WANG, D. KOUTSIOULIS, H.-K. S. LEIROS, O. A. ANDERSEN, V. BOURIOTIS, E. HOUGH and P. HEIKINHEIMO (2007) , J. Mol. Biol. 366, 1318–1331
3. Impact of Protein Flexibility on Hydride- Transfer Parameters in Thermophilic and Psychrophilic Alcohol Dehydrogenases” Z.X. LIANG, I. TSIGOS, V. BOURIOTIS, and J. P. KLINMAN (2004), JACS: 126, 9500-9501
One main defensive mechanism of bacterial pathogens is related to the modification of peptidoglycan backbone structure via either O -acetylation occurring on the C-6 hydroxyl moiety of the N -acetylmuramic acid residue or the de- N -acetylation of the N -acetylglucosamine residues. These modifications prevent binding of lysozyme and subsequent hydrolysis of the polysaccharide substrate.
Models of peptidoglycan O-acetylation
Two models of peptidoglycan O -acetylation have been proposed. The first mechanism involves a single protein which performs both the transport of acetate across the membrane and its transfer to peptidoglycan. oatA was the first known peptidoglycan O -acetyltransferase gene identified in Staphylococcus aureus. The second mechanism involves two proteins, one for acetate transport across the membrane and the other for catalyzing its transfer to MurNAc. The proteins involved in acetate transport have not been identified. Although experimental proof is lacking, there are several candidate genes encoding O -acetyltransferases (designated Pat) which are unrelated to OatA. Interestingly, irrespective of the species, the pat genes are always clustered on the chromosome with one or two ape genes, the products of which have O -acetylpeptidoglycan esterase activity capable of removing O -acetyl groups from peptidoglycan. At least 17 species contain a pat/ape gene cluster including Neisseria meningitis, Neisseria gonorrhoeae, H. pylori, B. anthracis and B. cereus.
The objective of this project is the biochemical characterization and elucidation of the biological role of the enzymes involved in the pat/ape clusters from B. anthracis. These enzymes may also serve as potential targets for the development of a new class of antibiotics specific for important human pathogenic bacteria.