Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution

Thermoanaerobacterium thermosulfurigenes cyclodextrin glucanotransferase primarily catalyses the formation of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. This enzyme also possesses unusually high hydrolytic activity as a side reaction, thought to be due to partial retention of ancestral enzyme function. This side reaction is undesirable, since it produces short saccharides that are responsible for the breakdown of the cyclodextrins formed, thus limiting the yield of cyclodextrins produced. To reduce the competing hydrolysis reaction, while maintaining the cyclization activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone PCR. Mutations in two residues, Ser-77 and Trp-239, on the outer region of the active site, lowered the hydrolytic activity up to 15-fold with retention of cyclization activity. In contrast, mutations within the active site could not lower hydrolytic rates, indicating an evolutionary optimized role for cyclodextrin formation by residues within this region. The crystal structure of the most effective mutant, S77P, showed no alterations to the peptide backbone. However, subtle conformational changes to the side chains of active-site residues had occurred, which may explain the increased cyclization/hydrolysis ratio. This indicates that secondary effects of mutations located on the outer regions of the catalytic site are required to lower the rates of competing side reactions, while maintaining the primary catalytic function. Subsequent functional analysis of various glucanotransferases from the superfamily of glycoside hydrolases also suggests a gradual evolutionary progression of these enzymes from a common ‘intermediate-like’ ancestor towards specific transglycosylation activity.

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Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

Biochemical Journal ◽

10.1042/bj3470865 ◽

2000 ◽

Vol 347 (3) ◽

pp. 865-873 ◽

Cited By ~ 5

Author(s):

Patricia NTARIMA ◽

Wim NERINCKX ◽

Klaus KLARSKOV ◽

Bart DEVREESE ◽

Mahalingeshwara K. BHAT ◽

...

Keyword(s):

Active Site ◽

Glycoside Hydrolase ◽

Glycoside Hydrolases ◽

Thermomyces Lanuginosus ◽

Glycoside Hydrolase Family ◽

Active Site Residues ◽

X Ray ◽

Order Of Magnitude ◽

Site Markers ◽

Hydrolase Family

A series of Ω-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant to electrophilic attack of active-site carboxyl residues, glycoside hydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. The apparent inactivation and association constants (ki, 1/Ki) are one order of magnitude higher for the xylobiose and xylotriose derivatives. The effects of the aglycone chain length can clearly be described. Xylobiose and n-alkyl β-D-xylopyranosides are competitive ligands and provide protection against inactivation. MS measurements showed 1:1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu86 as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl β-D-xylopyranoside was used, whereas the acid/base catalyst Glu178 was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu86 and Glu177 in T. reesei Xyn II are similarly modified, confirming earlier X-ray crystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996) Biochemistry 35, 9617-9624]. The inability of the Ω-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.

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Mutational Analysis of Active Site Residues Essential for Sensing of Organic Hydroperoxides by Bacillus subtilis OhrR

Journal of Bacteriology ◽

10.1128/jb.00879-07 ◽

2007 ◽

Vol 189 (19) ◽

pp. 7069-7076 ◽

Cited By ~ 16

Author(s):

Sumarin Soonsanga ◽

Mayuree Fuangthong ◽

John D. Helmann

Keyword(s):

Hydrogen Bond ◽

Bacillus Subtilis ◽

Conformational Changes ◽

Active Site ◽

Mutational Analysis ◽

High Sensitivity ◽

Oxidative Modification ◽

Active Site Residues

ABSTRACT Bacillus subtilis OhrR is the prototype for the one-Cys family of organic peroxide-sensing regulatory proteins. Mutational analyses indicate that the high sensitivity of the active site cysteine (C15) to peroxidation requires three Tyr residues. Y29 and Y40 from the opposing subunit of the functional dimer hydrogen bond with the reactive Cys thiolate, and substitutions at these positions reduce or eliminate the ability of OhrR to respond to organic peroxides. Y19 is also critical for peroxide sensing, and the Ala substitution mutant (OhrR Y19A) is less susceptible to oxidation at the active site C15 in vivo. The Y19A protein also displays decreased sensitivity to peroxide-mediated oxidation in vitro. Y19 is in van der Waals contact with two residues critical for protein function, F16 and R23. The latter residue makes critical contact with the DNA backbone in the OhrR-operator complex. These results indicate that the high sensitivity of the OhrR C15 residue to oxidation requires interactions with the opposed Tyr residues. Oxidative modification of C15 likely disrupts the C15-Y29′-Y40′ hydrogen bond network and thereby initiates conformational changes that reduce the ability of OhrR to bind to its operator site.

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Resistance from Afar: Distal Mutation V36M Allosterically Modulates the Active Site to Accentuate Drug Resistance in HCV NS3/4A Protease

10.1101/452284 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ayşegül Özen ◽

Kuan-Hung Lin ◽

Keith P Romano ◽

Davide Tavella ◽

Alicia Newton ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Active Site ◽

Md Simulations ◽

Drug Susceptibility ◽

Active Site Residues ◽

Allosteric Coupling ◽

Direct Acting ◽

Distal Mutation ◽

Active Site Mutations

AbstractHepatitis C virus rapidly evolves, conferring resistance to direct acting antivirals. While resistance via active site mutations in the viral NS3/4A protease has been well characterized, the mechanism for resistance of non-active site mutations is unclear. R155K and V36M often co-evolve and while R155K alters the electrostatic network at the binding site, V36M is more than 13 Å away. In this study the mechanism by which V36M confers resistance, in the context of R155K, is elucidated with drug susceptibility assays, crystal structures, and molecular dynamics (MD) simulations for three protease inhibitors: telaprevir, boceprevir and danoprevir. The R155K and R155K/V36M crystal structures differ in the α-2 helix and E2 strand near the active site, with alternative conformations at M36 and side chains of active site residues D168 and R123, revealing an allosteric coupling, which persists dynamically in MD simulations, between the distal mutation and the active site. This allosteric modulation validates the network hypothesis and elucidates how distal mutations confer resistance through propagation of conformational changes to the active site.

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FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5968 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5968-5975 ◽

Cited By ~ 24

Author(s):

C M Alarcón ◽

J Heitman

Keyword(s):

Saccharomyces Cerevisiae ◽

Conformational Changes ◽

Active Site ◽

Feedback Inhibition ◽

Immunosuppressive Drugs ◽

Intermediate Step ◽

Active Site Residues ◽

Peptidyl Prolyl Isomerase

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

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Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

Journal of Biological Chemistry ◽

10.1074/jbc.m110.211649 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16197-16207 ◽

Cited By ~ 26

Author(s):

Sebastian Reichau ◽

Wanting Jiao ◽

Scott R. Walker ◽

Richard D. Hutton ◽

Edward N. Baker ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Conformational Changes ◽

Active Site ◽

Catalytic Mechanism ◽

Condensation Reaction ◽

Shikimate Pathway ◽

Enzyme Reaction ◽

Multidrug Resistant ◽

Active Site Residues ◽

Site Water

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

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Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

F1000Research ◽

10.12688/f1000research.5147.1 ◽

2014 ◽

Vol 3 ◽

pp. 215 ◽

Cited By ~ 1

Author(s):

Hossein Gouran ◽

Sandeep Chakraborty ◽

Basuthkar J. Rao ◽

Bjarni Asgeirsson ◽

Abhaya M. Dandekar

Keyword(s):

Directed Evolution ◽

Active Site ◽

Xylella Fastidiosa ◽

Catalytic Efficiency ◽

Resistance Mechanisms ◽

Loss Of Function ◽

Active Site Residues ◽

Protein Protein Interaction ◽

Moonlighting Functions

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors ofXylella fastidiosa(LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we presentin vivovalidation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein fromXanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providingin silicovalidation before proceeding to the laboriousin vivowork. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

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Structural and functional roles of dynamically correlated residues in thymidylate kinase

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318002267 ◽

2018 ◽

Vol 74 (4) ◽

pp. 341-354 ◽

Cited By ~ 2

Author(s):

Santosh Kumar Chaudhary ◽

Jeyaraman Jeyakanthan ◽

Kanagaraj Sekar

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Active Site ◽

Bound States ◽

Catalytic Mechanism ◽

Phosphoryl Transfer ◽

Active Site Residues ◽

Thymidylate Kinase ◽

Binary And Ternary Complexes ◽

Insight Into

Thymidylate kinase is an important enzyme in DNA synthesis. It catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, with ATP as the preferred phosphoryl donor, in the presence of Mg2+. In this study, the dynamics of the active site and the communication paths between the substrates, ATP and TMP, are reported for thymidylate kinase fromThermus thermophilus. Conformational changes upon ligand binding and the path for communication between the substrates and the protein are important in understanding the catalytic mechanism of the enzyme. High-resolution X-ray crystal structures of thymidylate kinase in apo and ligand-bound states were solved. This is the first report of structures of binary and ternary complexes of thymidylate kinase with its natural substrates ATP and ATP–TMP, respectively. Distinct conformations of the active-site residues, the P-loop and the LID region observed in the apo and ligand-bound structures revealed that their concerted motion is required for the binding and proper positioning of the substrate TMP. Structural analyses provide an insight into the mode of substrate binding at the active site. The residues involved in communication between the substrates were identified through network analysis using molecular-dynamics simulations. The residues identified showed high sequence conservation across species. Biochemical analyses show that mutations of these residues either resulted in a loss of activity or affected the thermal stability of the protein. Further, molecular-dynamics analyses of mutants suggest that the proper positioning of TMP is important for catalysis. These data also provide an insight into the phosphoryl-transfer mechanism.

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Clinical Variants of the Native Class D β-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01277-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6155-6164 ◽

Cited By ~ 14

Author(s):

Emma C. Schroder ◽

Zachary L. Klamer ◽

Aysegul Saral ◽

Kyle A. Sugg ◽

Cynthia M. June ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Active Site ◽

Hydrolytic Activity ◽

Single Amino Acid ◽

Kinetic Properties ◽

Active Site Residues ◽

Content Type ◽

Class D ◽

Clinical Variants ◽

Dynamics Simulations

ABSTRACTThe threat posed by the chromosomally encoded class D β-lactamase ofAcinetobacter baumannii(OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of β-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of β-lactams against this troublesome pathogen.

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Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

PLoS ONE ◽

10.1371/journal.pone.0080448 ◽

2013 ◽

Vol 8 (11) ◽

pp. e80448 ◽

Cited By ~ 6

Author(s):

Takuji Oyama ◽

George E. Schmitz ◽

Dylan Dodd ◽

Yejun Han ◽

Alanna Burnett ◽

...

Keyword(s):

Active Site ◽

Glycoside Hydrolases ◽

Active Site Residues

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Energetic Control of Redox-Active Polymers Towards Safe Organic Bioelectronic Materials

10.26434/chemrxiv.11366186.v1 ◽

2019 ◽

Author(s):

Alexander Giovannitti ◽

Reem B. Rashid ◽

Quentin Thiburce ◽

Bryan D. Paulsen ◽

Camila Cendra ◽

...

Keyword(s):

Molecular Oxygen ◽

Organic Semiconductors ◽

Side Reaction ◽

Semiconducting Polymers ◽

Side Reactions ◽

Redox Active ◽

Donor Acceptor ◽

Electrochemical Devices ◽

Biological Environment ◽

Device Operation

Avoiding faradaic side reactions during the operation of electrochemical devices is important to enhance the device stability, to achieve low power consumption, and to prevent the formation of reactive side‑products. This is particularly important for bioelectronic devices which are designed to operate in biological systems. While redox‑active materials based on conducting and semiconducting polymers represent an exciting class of materials for bioelectronic devices, they are susceptible to electrochemical side‑reactions with molecular oxygen during device operation. We show that this electrochemical side reaction yields hydrogen peroxide (H2O2), a reactive side‑product, which may be harmful to the local biological environment and may also accelerate device degradation. We report a design strategy for the development of redox-active organic semiconductors based on donor-acceptor copolymers that prevent the formation of H2O2 during device operation. This study elucidates the previously overlooked side-reactions between redox-active conjugated polymers and molecular oxygen in electrochemical devices for bioelectronics, which is critical for the operation of electrolyte‑gated devices in application-relevant environments.

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