STRUCTURE/FUNCTION AND ENGINEERING OF CARBOHYDRATE ACTIVE ENZYMES. Applications in biocatalysis.

Project PI: Dr. Antoni Planas

Carbohydrate Active Enzymes (CAZymes) involved in the biosynthesis, degradation and modification of glycans and glycoconjugates are classified in five large families: glycosyl hydrolases or glycosidases (GH), glycosyltransferases (GT), carbohydrate esterases (CE), polysaccharide lyases (PL), carbohydrate esterases (CE) and auxiliary activities (AA). Targeting diverse enzyme families, we study the mechanistic aspects of enzyme catalysis and the structural determinants of substrate specificity aimed at: i) Unravel structure/function relationships and evaluate target enzymes as potential therapeutic targets. ii) Enzyme engineering, either by knowledge-based design or directed evolution strategies, to develop applications in biocatalysis addressing the synthesis of relevant natural and non-natural glycostructures for different application fields.

Main topics include:
A) De-N-acetylation of structural polysaccharides by peptidoglycan and chitin deacetylases
B) Glycosynthesis by engineered glycosyl hydrolases as glycosynthases and transglycosidases
C) Structure-function of glycosyltransferases as therapeutic targets

a) De-N-acetylation of structural polysaccharides by peptidoglycan and chitin deacetylases.

Deacetylation of their own cell wall polysaccharides is a strategy used by pathogenic bacteria and fungi to evade the host immune responses at initial stages of infection. Pathogenic bacteria utilize acetylation (6-O-acetylation of MurNAc) and deacetylation (2-N-deacetylation of GlcNAc and/or MurNAc residues) of their cell wall peptidoglycan (PGN) to evade detection by the innate immune system. Likewise, plant pathogenic fungi partially deacetylate their cell wall chitin to be resistant to degradation by plant chitinases or deacetylate the released chitooligosaccharides (COS) to escape recognition by chitin receptors and evade the plant immune responses. Peptidoglycan and chitin deacetylases are members of family 4 carbohydrate esterases (CE4 enzymes) which operate by a metal-assisted general acid/base catalytic mechanism. We are interested in understanding the structural bases of substrate specificity by CE4 enzymes, their mechanism of action and biological functions, as well as developing engineered variants for biocatalysis.

Chitin deacetylases. CDAs exhibit diverse deacetylation patterns, reflecting different specificities and pattern recognition on their substrates, being processive, distributive or deacetylating a single position on chitooligosaccharides (COS). Because of the influence of different deacetylation patterns in signaling events (i.e. pathogenic fungi-host interactions), the availability of a panel of CDAs with defined specificity will provide sequence-defined partially deacetylated COS with broad applications in biomedicine and biotechnology. We are identifying and characterizing new CDAs and engineering their specificity with regard to the deacetylation pattern with the aim of expanding the portfolio of enzymes for biocatalysis.

A) Chitin deacetylase from Vibrio cholera (VcCDA).The subsite capping model guiding loops engineering for novel specificities. B) Metal-assisted general acid/base catalytic mechanism.. C) HTS assay for screening directed evolution libraries.

Peptidoglycan deacetylases. Remodeling of bacterial cell wall peptidoglycan (PGN) has critical functions in PGN maturation, elongation, septation, turnover, and recycling. Modifications of PGN represent an important strategy for pathogenic bacteria to evade innate immunity of the host and control autolysis. PGN GlcNAc and MurNAc deacetylases have mutually exclusive specificities, but currently no sequence or structural signatures can be assigned to each enzyme class to predict specificity and function. We investigate the structure/funtion and evolutionary relationships of PGN deacetylases and related CE4 enzymes to unravel the structural determinants of substrate specificity and evaluate them as potential therapeutic targets for the design of antimicrobials.

Peptidoglycan MurNAc deacetylases from Bacillus subtillis. Two Pda subfamilies with different specificities with regard to the stem peptide substitution of the glycan chain. PdaA involved in sporulation, PdaC with unidentified biological function.

Selected publications:

Carbohydrate de-N-acetylases acting on structural polysaccharides and glycoconjugates.
S. Pascual, A Planas.
Current Opinion in Chemical Biology 61, 9–18 (2021). Abstract. https://doi.org/10.1016/j.cbpa.2020.09.003

Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the peptidoglycan deacetylase PdaC from Bacillus subtilis.
L. Grifoll-Romero, M.A. Sainz-Polo, D. Albesa-Jové, M.E. Guerin, X. Biarnés, A. Planas.
Journal of Biological Chemistry 294, 19066-80 (2019). Abstract. http://dx.doi.org/10.1074/jbc.RA119.009510

Screening assay for directed evolution of chitin deacetylases. Application to Vibrio cholerae deacetylase mutant libraries for engineered specificity.
S. Pascual, A. Planas.
Analytical Chemistry 90, 10654–10658 (2018). Abstract. http://dx.doi.org/10.1021/acs.analchem.8b02729

Chitin deacetylases: structures, specificities, and biotech applications.
L. Grifoll, S. Pascual, H. Aragunde, X. Biarnés, A. Planas.
Polymers 10, 352 (2018). Abstract. https://doi.org/10.3390/polym10040352

Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases.
S.N. Hamer, S. Cord-Landwehr, X. Biarnés, A. Planas, H. Waegeman, B.M. Moerschbacher, S. Kolkenbrock.
Scientific Reports 5, 8716 (2015). Abstract. http://dx.doi.org/10.1038/srep08716

Structural basis of chitin oligosaccharide deacetylation.
E. Andrés, D. Albesa-Jové, X. Biarnés, B. M. Moerschbacher, M. E. Guerin, A. Planas.
Angewandte Chemie Int. Ed. 53, 6882–6887 (2014). http://dx.doi.org/10.1002/anie.201400220

b) Glycosynthesis by engineered glycosyl hydrolases as glycosynthases and transglycosidases

Synthetic tools to access natural and non-natural glycosides and glycoconjugates is a central issue in glycochemistry and glycobiology. Both for industrial applications in diverse fields and biological research in functional glycomic studies, there is the need of efficient synthetic approaches for structurally defined oligosaccharides and glycoconjugates. In contrast to conventional chemical synthesis that requires complex and tedious protection/deprotection manipulations and activation strategies, enzymatic glycosylation offers many advantages, since enzymes provide selectivity, acting with perfect control of the anomeric configuration and with high regioselectivity without the need of protecting groups under mild conditions.

Glycosidases are the natural hydrolyzing enzymes that catalyze the breakdown of glycans and glycoconjugates. Taking advantage of their specificity, the goal is to reengineer the mechanism towards synthetic abilities: a) the “Glycosynthase technology” by engineering the catalytic machinery and using activated glycosyl donors, and b) modulate the transglycosylation to hydrolysis ratio towards glycoside bond formation. Together with other groups, we pioneered the development of the glycosynthase technology (Withers et al., Planas et al., Moracci et al.) and applied it to diverse GH families to enzymatically synthesize glycans and glycoconjugates with defined structures in high yields.

     When enzymes do it better: enzymatic glycosylation methods.
     A. Planas, M. Faijes, V. Codera.
     In Carbohydrates Chemistry: State-of-the-art and challenges for drug development.
     (L. Cipolla, Ed.), Imperial College Press, London, July 2015, pp.215-245. ISBN: 9781783267194

Recent examples on glycosynthase and transglycosylase approaches:

a) Artificial sequence-defined polysaccharides by engineered glycosynthases
Sequence control is one of the great remaining problems in polysaccharide synthesis. Nature creates polysaccharides often with a very high degree of control over sequence, leading to highly specific biological activities. We envision the glycosynthase technology as a potential tool for the efficient synthesis of functionalized polysaccharides: simple glycosyl donors prepared with the specific modifications can be accepted and polymerized by the glycosynthase enzyme to produce sequence-defined and modified polysaccharides. We have prepared mixed-linked 1,3-1,4-glucans and alternating 6’-substituted celluloses and the technology will be further extended to more complex patterned glycans for graphting bioactive molecules.

Sequence-defined polysaccharides by glycosynthase-catalyzed polymerization. (Left) Mixed linked β-1,3-1,4-glucans (MW up to 30 kDa); (Right) Artificial functionalized cellulose (MW 5 kDa).

Selected publications:

Functionalized celluloses with regular substitution pattern by glycosynthase-catalyzed polymerization.
V. Codera, K.J. Edgar, M. Faijes, A. Planas.
Biomacromolecules, 17(4),1272-9 (2016). Abstracthttp://dx.doi.org/10.1021/acs.biomac.5b01453

Carbohydrate Binding Module assisting glycosynthase-catalyzed polymerizations.
V. Codera, H. J. Gilbert, M. Faijes, A.Planas.
Biochemical Journal 470, 15-22 (2015). Abstracthttp://dx.doi.org/10.1042/BJ20150420

Artificial Mixed-Linked β-Glucans Produced by Glycosynthase-Catalyzed Polymerization: Tuning Morphology and Degree of Polymerization.
X. Pérez, M. Faijes, A. Planas.
Biomacromolecules 12, 494–501 (2011). https://doi.org/10.1021/bm1013537

b) Chitooligosaccharides by engineered chitinases as glycosynthases
Chitinases (Chi) are glycoside hydrolases that catalyze the hydrolysis of chitin and chitosans to short oligosaccharides (COS). Family 18 chitinases (GH18) operate by a double displacement mechanism with substrate assisted catalysis. We are engineering GH18 chitinases to introduce synthase activity for the controlled polymerization of COS with the aim of producing polymeric sequence-defined chitosans and their evaluation as novel biomaterials.

Enzyme-catalyzed polymerization by engineered GH18 chitinases as glycosynthases. Chitin and chitosan oligo/polysaccharides.

Selected publications: 

Auxiliary active site mutations enhance the glycosynthase activity of a GH18 chitinase for polymerization of chitooligosaccharides.
C. Alsina, E. Sancho-Vaello, A. Aranda-Martínez, M. Faijes, A. Planas.
Carbohydrate Polymers 252, 117121 (2021). Abstracthttps://doi.org/10.1016/j.carbpol.2020.117121

Glycosynthase-type GH18 mutant chitinases at the assisting catalytic residue for polymerization of chitooligosaccharides.
C. Alsina, M. Faijes, A. Planas.
Carbohydrate Research 478, 1-9 (2019). Abstracthttps://doi.org/10.1016/j.carres.2019.04.001

c) Human milk oligosaccharides (HMOs). Engineered transglycosidases for enzymatic HMO synthesis
Human milk oligosaccharides (HMO) furnish breast-fed infants with a number of health benefits as prebiotics and antimicrobial agents as well as exerting immunomodulation effects. Their unique composition differing from other mammal’s milk drives active research to synthesize and produce the main HMO structures as supplements for infant formula milks. Lacto-N-tetraose is one of the main core structures that is further extended and functionalized with fucosyl and/or sialyl units at different positions. Using hydrolases involved in HMO catabolism by Bifidobacteria, we aim at engineering GH20 glycosidases into synthetic enzymes by modulating the transglycosylation to hydrolysis ratio towards efficient biocatalysts.

Lacto-N-tetraose synthesis by transglycosylating mutants of  GH20 lacto-N-biosidase

Selected publications: 

Transglycosylation activity of engineered Bifidobacterium lacto-N-biosidase mutants at donor subsites for lacto-N-tetraose synthesis.
M. Castejón-Vilatersana, M. Faijes, A. Planas.
International Journal of Molecular Sciences 22, 3230 (2021). Abstracthttps://doi.org/10.3390/ijms22063230

Enzymatic and cell factory approaches to the production of human milk oligosaccharides.
M. Faijes, M. Castejón, C. Val-Cid, A. Planas.
Biotechnology Advances 37, 667–697 (2019). Abstracthttps://doi.org/10.1016/j.biotechadv.2019.03.014

Structural-functional analysis reveals a specific domain organization in family GH20 hexosaminidases.
C. Val-Cid, X. Biarnés, M. Faijes, A. Planas.
PLOS One 10(5): e0128075 (2015). Abstracthttp://dx.doi.org/10.1371/journal.pone.0128075

C) Structure-function of glycosyltransferases as therapeutic targets.

Glycoglycerolipids (GGL) are found in plant chloroplasts and cyanobacteria (galactolipids) as membrane components associated with photosynthetic tissues. Similar glycolipids with a wider structural diversity are widespread in Gram-positive bacteria where they serve as lipid anchors for lipotheichoic acids. Mycoplasmas, prokaryotes devoid of cell wall, are particularly rich in glycoglycerolipids with a fundamental role in membrane stability and fluidity. Since GGL are absent in animal cells, the enzymes involved in membrane GGL biosynthesis in pathogenic mycoplasmas are potential therapeutic targets.

We identified and characterized a Mycoplasma genitalium glycosyltransferase, GT M517, that is a membrane-associated and sequentially acting GT2 producing monoglycosyl and diglycosyldiacylglycerols (gluco- or galacto-lipids) , regulated by anionic lipids and essential for mycoplasma viability. The modelled structure predicts a monotopic association with the membrane, sustained by mutational studies. Mycoplasma growth is inhibited by GT MG517 inhibitors and a drug discovery program has been initiated to validating the GT as an antimicrobial target against mycoplasma infections.

GT M517 from Mycoplasma genitalium. Sequential glycolipid synthase (GT2), membrane-associated and activated by anionic lipids. Target for drug design against mycoplasma infections.

Selected publications: 

Essential mycoplasma glycolipid synthase adheres to the cell membrane by means of an amphipathic helix.
J. Romero-García, X. Biarnés, A. Planas.
Scientific Reports 9, 7085 (2019). Abstracthttps://doi.org/10.1038/s41598-019-42970-9

Structure-Function Features of a Mycoplasma Glycolipid Synthase Derived from Structural Data Integration, Molecular Simulations, and Mutational Analysis.
J. Romero-García, C. Francisco, X. Biarnés, A. Planas.
PLOS One 8(12), e81990 (2013). http://dx.doi.org/10.1371/journal.pone.0081990

Expression and characterization of a Mycoplasma genitalium glycosyltransferase in membrane glycolipid biosynthesis. Potential target against mycoplasma infections.
E. Andrés, N. Martínez, A. Planas.
Journal of Biological Chemistry 286, 35367–35379 (2011). https://doi.org/10.1074/jbc.M110.214148