Molecular Dynamics Simulations Provide Insights into Structure and Function of Amadoriase Enzymes
Background. Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes.
Objective. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnel, ligand binding residues and ligand binding energies.
Methods. We used a combination of homology modeling and molecular dynamics simulations to provide insights into the structural difference between the five enzymes of the FAOX family.
Results. We first predicted the structure of the N1-1-FAOD and PnFPOX enzymes using homology modelling. Then, we used these models and the experimental crystal structures of Amadoriase I, Amadoriase II and FPOX-E to run extensive molecular dynamics simulations in order to compare the structures of these FAOX enzymes and assess their relevant interactions with two relevant ligands, f-val and f-lys.Conclusions. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels.
C. Weykamp et al., “A review of the challenge in measuring hemoglobin A1c”, J. Diabetes Sci. Technol., vol. 3, no. 3, pp. 439–445, 2009. doi: 10.1177/193229680900300306
W.G. John, “Haemoglobin A1c: analysis and standardisation”, Clin. Chem. Lab. Med., vol. 41, no. 3, pp. 1199–1212, 2003. doi: 10.1515/CCLM.2003.184
L. Liu et al., “Direct enzymatic assay for %HbA1c in human whole blood samples”, Clin. Biochem., vol. 41, no. 7-8, pp. 576–583, 2008. doi: 10.1016/j.clinbiochem.2008.01.013
S. Miura et al., “Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis”, Protein Eng. Des. Sel., vol. 21, no. 4, pp. 233–239, 2008. doi: 10.1093/protein/gzm047
S. Kim et al., “Cumulative effect of amino acid substitution for the development of fructosyl valine-specific fructosyl amine oxidase”, Enzyme Microb. Technol., vol. 44, no. 1, pp. 52–56, 2009. doi: 10.1016/j.enzmictec.2008.09.001
S. Miura et al., “Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis”, Biotechnol. Lett., vol. 28, pp. 1895–1900, 2006. doi: 10.1007/s10529-006-9173-9
S. Kim et al., “Engineering of dye-mediated dehydrogenase property of fructosyl amino acid oxidases by site-directed mutagenesis studies of its putative proton relay system”, Biotechnol. Lett., vol. 32, no. 8, pp. 1123–1129, 2010. doi: 10.1007/ s10529-010-0267-z
S. Kim et al., “Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions”, Biotechnol. Lett., vol. 34, no. 3, pp. 491–497, 2012. doi: 10.1007/s10529-011-0787-1
C. Mennella et al., “Substrate specificity of amadoriase I from Aspergillus fumigatus”, Ann. N. Y. Acad. Sci., vol. 1043, pp. 837–844, 2005. doi: 10.1196/annals.1333.096
X. Wu et al., “Kinetic studies, mechanism, and substrate specificity of amadoriase I from Aspergillus sp.”, Biochemistry, vol. 40, no. 43, pp. 12886–95, 2001. doi: 10.1021/Bi011244e
Y. Qian et al., “Loop engineering of amadoriase II and mutational cooperativity”, Appl. Microbiol. Biotechnol., vol. 97, no. 19, pp. 8599–8607, 2013. doi: 10.1007/s00253-013-4705-4
J. Zheng et al., “Engineered amadoriase II exhibiting expanded substrate range”, Appl. Microbiol. Biotechnol., vol. 86, no. 2, pp. 607–613, 2010. doi: 10.1007/s00253-009-2319-7
F. Collard et al., “Crystal structure of the deglycating enzyme fructosamine oxidase (amadoriase II)”, J. Biol. Chem., vol. 283, no. 40, pp. 27007–27016, 2008. doi: 10.1074/jbc.M804885200
W. Gan et al., “Structural basis of the substrate specificity of the FPOD/FAOD family revealed by fructosyl peptide oxidase from Eupenicillium terrenum”, Acta Crystallogr. Sect. F, Struct. Biol. Commun., vol. 71, pp. 381–387, 2015. doi: 10.1107/ s2053230x15003921
F. Rigoldi et al., “Crystal structure of the deglycating enzyme amadoriase i in its free form and substrate-bound complex”, Proteins, vol. 84, pp. 744–758, 2016. doi: 10.1002/prot.25015
A. Masic et al., “Osmotic pressure induced tensile forces in tendon collagen”, Nat. Commun., vol. 6, pp. 1–8, 2015. doi: 10.1038/ ncomms6942
Z. Qin and M.J. Buehler, “Impact tolerance in mussel thread networks by heterogeneous material distribution”, Nat. Commun., vol. 4, p. 2187, 2013. doi: 10.1038/ncomms3187
A. Gautieri et al.. “Vesentini, Age- and diabetes-related nonenzymatic crosslinks in collagen fibrils: Candidate amino acids involved in Advanced Glycation End-products”, Matrix Biol., vol. 34, pp. 89–95, 2013. doi: 10.1016/j.matbio.2013.09.004
A. Gautieri et al., “Modeling and measuring visco-elastic properties: From collagen molecules to collagen fibrils”, Int. J. Non. Linear. Mech., vol. 56, pp. 25–33, 2013. doi: 10.1016/j.ijnonlinmec.2013.03.012
A. Gautieri et al., “How to predict diffusion of medium-sized molecules in polymer matrices. From atomistic to coarse grain simulations”, J. Mol. Model., vol. 16, no. 12, 1845–1851, 2010. doi: 10.1007/s00894-010-0687-7
O. Tokareva et al., “Structure-function-property-design interplay in biopolymers: Spider silk”, Acta Biomater., vol. 10, no. 4, pp. 1612–1626, 2014. doi: 10.1016/j.actbio.2013.08.020
M. Takeuchi et al., “Immunological detection of fructose-derived advanced glycation end-products”, Lab. Invest., vol. 90, pp. 1117–1127, 2010. doi: 10.1038/labinvest.2010.62
M.I. Solar et al., “Composite materials: Taking a leaf from nature’s book”, Nat. Nanotechnol., vol. 7, pp. 417–419, 2012. doi: 10.1038/nnano.2012.86
S. Kim et al., “Motif-based search for a novel fructosyl peptide oxidase from genome databases”, Biotechnol. Bioeng., vol. 106, no. 3, pp. 358–366, 2010. doi: 10.1002/bit.22710
K. Hirokawa et al., “Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein”, Biochem. Biophys. Res. Commun., vol. 311, no. 1, pp. 104–111, 2003. doi: 10.1016/j.bbrc.2003.09.169
Z. Lin and J. Zheng, “Occurrence, characteristics, and applications of fructosyl amine oxidases (amadoriases)”, Appl. Microbiol. Biotechnol., vol. 86, no. 6, pp. 1613–9, 2010. doi: 10.1007/s00253-010-2523-5
M.A. Larkin et al., “Clustal W and Clustal X version 2.0”, Bioinformatics, vol. 23, no. 21, pp. 2947–2948, 2007. doi: 10.1093/ bioinformatics/btm404
N. Saitou and M. Nei, “The neighbor-joining method: a new method for reconstructing phylogenetic trees”, Mol. Biol. Evol., vol. 4, no. 4, pp. 406–425, 1987.
M. Kimura, “A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences”, J. Mol. Evol., vol. 16, no. 2, pp. 111–120, 1980.
M. Takahashi et al., “Molecular cloning and expression of amadoriase isoenzyme (fructosyl amine:oxygen oxidoreductase, EC 1.5.3) from Aspergillus fumigatus”, J. Biol. Chem., vol. 272, no. 19, pp. 12505–12507, 1997. doi: 10.1074/ jbc.272.19.12505
X. L. Wu et al., “Cloning of amadoriase I isoenzyme from Aspergillus sp.: Evidence of FAD covalently linked to Cys342”, Biochemistry, vol. 39, no. 6, pp. 1515–1521, 2000. doi: 10.1021/bi992031g
M. Takahashi et al., “Isolation, purification, and characterization of amadoriase isoenzymes (fructosyl amine-oxygen oxidoreductase EC 1.5.3) from Aspergillus sp”, J. Biol. Chem., vol. 272, no. 6, pp. 3437–3443, 1997. doi: 10.1074/jbc.272.6.3437
S. Ferri et al., “Engineering fructosyl peptide oxidase to improve activity toward the fructosyl hexapeptide standard for HbA1c measurement”, Mol. Biotechnol., vol. 54, no. 3, pp. 939–943, 2013. doi: 10.1007/s12033-012-9644-2
F. Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega”, Mol. Syst. Biol., vol. 7, no. 1, p. 539, 2011. doi: 10.1038/msb.2011.75
H. McWilliam et al., “Analysis tool web services from the EMBL-EBI”, Nucleic Acids Res., vol. 41, Web Server issue, pp. 597–600, 2013. doi: 10.1093/nar/gkt376
W. Li et al., “The EMBL-EBI bioinformatics web and programmatic tools framework”, Nucleic Acids Res., vol. 43, no. W1, pp. W580-4, 2015. doi: 10.1093/nar/gkv279
X. Robert and P. Gouet, “Deciphering key features in protein structures with the new ENDscript server”, Nucleic Acids Res., vol. 42, no. W1, pp. W320–W324, 2014. doi: 10.1093/nar/gku316
S. Ferri et al., “Review of fructosyl amino acid oxidase engineering research: a glimpse into the future of hemoglobin A1c biosensing”, J. Diabetes Sci. Technol., vol. 3, no. 3, pp. 585–592, 2009. doi: 10.1177/193229680900300324
K. Hirokawa et al., “Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi”, Arch. Microbiol., vol. 180, no. 3, pp. 227–231, 2003. doi: 10.1007/s00203-003-0584-x
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