Just an additional hydrogen bond can dramatically reduce the catalytic activity of Bacillus subtilis lipase A I12T mutant: An integration of computational modeling and experimental analysis

Understanding the structural basis and energetic property of hydrogen bonding and its effects on enzymatic activity is fundamentally important for the rational design of specific enzymes with desired biological functions. In the current study, site-directed mutagenesis analysis preliminarily revealed that the amino acid substitution of Ile12 with Thr12 (I12T) dramatically reduced the hydrolytic activity of Bacillus subtilis lipase A. A further computational investigation proposed that the I12T mutation would establish a geometrically perfect hydrogen bond between the mutated Thr12 and catalytic Ser77 of lipase A, which considerably impaired the catalytic capability of lipase A through two distinct but complementary approaches: rigidizing the enzyme active site and lowering the nucleophilic ability of the catalytic residue Ser77. To verify this hypothesis, a homogenous mutation I12S serving as the control to the I12T mutation was created to examine the hydrogen bonding effect on enzymatic activity. It was found that the I12S mutant only suffered from a slight damage in its hydrolytic ability due to absence of the hydrogen bond originally present at the Thr12-Ser77 interface in the I12T mutant, which was further characterized systematically by quantum mechanics/molecular mechanics (QM/MM) modeling, atom-in-molecules (AIM) analysis and molecular dynamics (MD) simulation. It is suggested that the hydrogen bond arising from the I12T mutation in lipase A can considerably reduce the flexibility and mobility of the enzyme active site, thus impairing the catalytic activity of the lipase A I12T mutant remarkably; the activity loss can be, however, largely recovered by replacing Thr residue at the 12th position of I12T mutant with its analog Ser, which is chemically similar to Thr but cannot form effective hydrogen bonding with Ser77.

[1]  Peng Zhou,et al.  Gaussian process: an alternative approach for QSAM modeling of peptides , 2008, Amino Acids.

[2]  K. Hult,et al.  Amidases Have a Hydrogen Bond that Facilitates Nitrogen Inversion, but Esterases Have Not , 2011 .

[3]  S. F. Boys,et al.  The calculation of small molecular interactions by the differences of separate total energies. Some procedures with reduced errors , 1970 .

[4]  K. Sode,et al.  Mutational analysis of the oxygen-binding site of cholesterol oxidase and its impact on dye-mediated dehydrogenase activity , 2013 .

[5]  B. Barquera,et al.  The Role of Glycine Residues 140 and 141 of Subunit B in the Functional Ubiquinone Binding Site of the Na+-pumping NADH:quinone Oxidoreductase from Vibrio cholerae* , 2012, The Journal of Biological Chemistry.

[6]  Peng Zhou,et al.  Fluorine Bonding - How Does It Work In Protein-Ligand Interactions? , 2009, J. Chem. Inf. Model..

[7]  Eric N. Jacobsen,et al.  Attractive noncovalent interactions in asymmetric catalysis: Links between enzymes and small molecule catalysts , 2010, Proceedings of the National Academy of Sciences.

[8]  T. K. Nandi,et al.  Conserved water-mediated H-bonding dynamics of catalytic His159 and Asp158: insight into a possible acid–base coupled mechanism in plant thiol protease , 2012, Journal of Molecular Modeling.

[9]  D. van der Spoel,et al.  GROMACS: A message-passing parallel molecular dynamics implementation , 1995 .

[10]  F. Tian,et al.  In silico quantitative prediction of peptides binding affinity to human MHC molecule: an intuitive quantitative structure–activity relationship approach , 2009, Amino Acids.

[11]  K. S. Kim,et al.  Catalytic role of enzymes: short strong H-bond-induced partial proton shuttles and charge redistributions. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Halogen-Ionic Bridges: Do They Exist in the Biomolecular World? , 2010, Journal of chemical theory and computation.

[13]  H. Berendsen,et al.  Interaction Models for Water in Relation to Protein Hydration , 1981 .

[14]  B. Dijkstra,et al.  The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase fold enzyme. , 2001, Journal of molecular biology.

[15]  M. Ortiz-Maldonado,et al.  Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis. , 2004, Biochemistry.

[16]  M. Challacombe,et al.  Rapid evaluation of atomic properties with mixed analytical/numerical integration , 1993 .

[17]  W. Goddard,et al.  UFF, a full periodic table force field for molecular mechanics and molecular dynamics simulations , 1992 .

[18]  Andrew C. Simmonett,et al.  Exploring the effects of H-bonding in synthetic analogues of nickel superoxide dismutase (Ni-SOD): experimental and theoretical implications for protection of the Ni-SCys bond. , 2010, Inorganic chemistry.

[19]  Thom Vreven,et al.  Geometry optimization with QM/MM, ONIOM, and other combined methods. I. Microiterations and constraints , 2003, J. Comput. Chem..

[20]  Keng-Ming Chang,et al.  Roles of amino acids in the Escherichia coli octaprenyl diphosphate synthase active site probed by structure-guided site-directed mutagenesis. , 2012, Biochemistry.

[21]  J. Åqvist,et al.  The catalytic power of ketosteroid isomerase investigated by computer simulation. , 2002, Biochemistry.

[22]  S. Shaik,et al.  Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzyme , 2004 .

[23]  Eamonn F. Healy,et al.  Development and use of quantum mechanical molecular models. 76. AM1: a new general purpose quantum mechanical molecular model , 1985 .

[24]  G. T. Marks,et al.  Short, strong hydrogen bonds on enzymes: NMR and mechanistic studies , 2002 .

[25]  A. J. Kirby,et al.  Enzyme Mechanisms, Models, and Mimics , 1996 .

[26]  Sason Shaik,et al.  Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes. , 2004, Chemical reviews.

[27]  J. Goodman,et al.  Hydrogen-bond stabilization in oxyanion holes: grand jeté to three dimensions. , 2012, Organic & biomolecular chemistry.

[28]  Chao Yang,et al.  Computational peptidology: a new and promising approach to therapeutic peptide design. , 2013, Current medicinal chemistry.

[29]  Chao Yang,et al.  What are the ideal properties for functional food peptides with antihypertensive effect? A computational peptidology approach. , 2013, Food chemistry.

[30]  Friedrich Biegler-König,et al.  Calculation of the average properties of atoms in molecules. II , 1982 .

[31]  Denis J. Evans,et al.  The Nose–Hoover thermostat , 1985 .

[32]  R. Wentzcovitch,et al.  Invariant molecular-dynamics approach to structural phase transitions. , 1991, Physical review. B, Condensed matter.

[33]  J. Cioslowski,et al.  A new robust algorithm for fully automated determination of attractor interaction lines in molecules , 1994 .

[34]  S. Boxer,et al.  Site-specific measurement of water dynamics in the substrate pocket of ketosteroid isomerase using time-resolved vibrational spectroscopy. , 2012, The journal of physical chemistry. B.

[35]  Jian Huang,et al.  Computational Peptidology , 2015, Methods in Molecular Biology.

[36]  B. Dijkstra,et al.  The Crystal Structure of Bacillus subtilis Lipase : A Minimal α/β Hydrolase Fold Enzyme , 2001 .

[37]  T. K. Nandi,et al.  Structural and Putative Functional Role of Conserved Water Molecular Cluster in the X-ray Structures of Plant Thiol Proteases: A Molecular Dynamics Simulation Study , 2012, Journal of Chemical Crystallography.

[38]  A. H. Wang,et al.  A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis. , 2008, The Biochemical journal.

[39]  P. Zhou,et al.  A Combination of Computational and Experimental Approaches to Investigate the Binding Behavior of B.sub Lipase A Mutants with Substrate pNPP , 2011, Molecular informatics.

[40]  G. Evans,et al.  Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase. , 2002, Biochemistry.

[41]  Satoko Hayashi,et al.  Atoms-in-molecules dual parameter analysis of weak to strong interactions: behaviors of electronic energy densities versus Laplacian of electron densities at bond critical points. , 2008, The journal of physical chemistry. A.

[42]  Alex V. Pickering,et al.  Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase. , 2013, Biochemistry.

[43]  F. Tian,et al.  Geometric characteristics of hydrogen bonds involving sulfur atoms in proteins , 2009, Proteins.

[44]  R. Bader,et al.  An Atoms‐In‐Molecules study of the genetically‐encoded amino acids: I. Effects of conformation and of tautomerization on geometric, atomic, and bond properties , 2000, Proteins.