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

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

Biochemical Journal ◽

10.1042/bj20080353 ◽

2008 ◽

Vol 413 (3) ◽

pp. 517-525 ◽

Cited By ~ 37

Author(s):

Ronan M. Kelly ◽

Hans Leemhuis ◽

Henriëtte J. Rozeboom ◽

Niels van Oosterwijk ◽

Bauke W. Dijkstra ◽

...

Keyword(s):

Directed Evolution ◽

Conformational Changes ◽

Active Site ◽

Outer Region ◽

Side Reaction ◽

Hydrolytic Activity ◽

Glycoside Hydrolases ◽

Side Reactions ◽

Cyclodextrin Glucanotransferase ◽

Active Site Residues

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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Faculty Opinions recommendation of 13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1000833.9459 ◽

2001 ◽

Author(s):

Hung-Wen Liu

Keyword(s):

13C Nmr ◽

Active Site ◽

Electrostatic Interactions ◽

Nmr Analysis ◽

Active Site Residues ◽

13C Nmr Analysis ◽

Uridine Nucleotides

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Development of Xanthine Based Inhibitors Targeting Phosphodiesterase 9A

Letters in Drug Design & Discovery ◽

10.2174/1570180813666161102125423 ◽

2017 ◽

Vol 14 (10) ◽

pp. 1122-1137 ◽

Cited By ~ 1

Author(s):

Nivedita Singh ◽

Parameswaran Saravanan ◽

M.S. Thakur ◽

Sanjukta Patra

Keyword(s):

Free Energy ◽

Active Site ◽

Signal Transduction Pathway ◽

Docking Study ◽

Primary Objective ◽

Cgmp Level ◽

Active Site Residues ◽

Aromatic Fragment ◽

Docking Approach ◽

Free Energy Of Binding

Background: Phosphodiesterases 9A (PDE9A) is one of the prominent regulating enzymes of the signal transduction pathway having highest catalytic affinity for second messenger, cGMP. When the cGMP level is lowered, an uncontrolled expression of PDE9A may lead to various neurodegenerative diseases. To regulate the catalytic activity of PDE9A, potent inhibitors are needed. Objective: The primary objective of the present study was to develop new xanthine based inhibitors targeting PDE9A. This study was an attempt to bring structural diversification in PDE9A inhibitor development because most of the existing inhibitors are constructed over pyrazolopyrimidinone scaffold. Methods: Manual designing and parallel molecular docking approach were used for the development of xanthine derivatives. In this study, N1, N3, N9 and C8 positions of xanthine scaffold were selected as substitution sites to design 200 new compounds. Reverse docking and pharmaceutical analyses were used for final validation of most promising compounds. Results: By keeping free energy of binding cut-off of -6.0 kcal/mol, 52 compounds were screened. The compounds with substitution at N1, N3 and C8 positions of xanthine showed good occupancy in PDE9A active site pocket with a significant interaction pattern. This was further validated by screening different factors such as free energy of binding, inhibition constant and interacting active site residues in the 5Å region. Substitution at C8 position with phenyl substituent determined the inhibition affinity of compounds towards PDE9A by establishing a strong hydrophobic - hydrophobic interaction. The alkyl chain at N1 position generated selectivity of compounds towards PDE9A. The aromatic fragment at N3 position increased the binding affinity of compounds. Thus, by comparative docking study, it was found that compound 39-42 formed selective interaction towards PDE9A over other members of the PDE superfamily. Conclusion: From the present study, N1, N3 and C8 positions of xanthine were concluded as the best sites for substitution for the generation of potent PDE9A inhibitors.

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An enzymatic activation of formaldehyde for nucleotide methylation

Nature Communications ◽

10.1038/s41467-021-24756-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Charles Bou-Nader ◽

Frederick W. Stull ◽

Ludovic Pecqueur ◽

Philippe Simon ◽

Vincent Guérineau ◽

...

Keyword(s):

Amino Acids ◽

Thymidylate Synthase ◽

Active Site ◽

De Novo ◽

Crystallographic Structure ◽

De Novo Synthesis ◽

Active Site Residues ◽

Enzymatic Activation ◽

Enzyme Cofactors ◽

Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)52245-6 ◽

1991 ◽

Vol 266 (4) ◽

pp. 2313-2319

Author(s):

R L Van Etten ◽

R Davidson ◽

P E Stevis ◽

H MacArthur ◽

D L Moore

Keyword(s):

Acid Phosphatase ◽

Active Site ◽

Prostatic Acid Phosphatase ◽

Active Site Residues ◽

Disulfide Bonding ◽

Reactive Surface ◽

Covalent Structure

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Identification of two active site residues in human angiotensin I-converting enzyme.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43897-5 ◽

1994 ◽

Vol 269 (47) ◽

pp. 29430-29434

Author(s):

T A Williams ◽

P Corvol ◽

F Soubrier

Keyword(s):

Active Site ◽

Converting Enzyme ◽

Active Site Residues ◽

Angiotensin I ◽

Angiotensin I Converting Enzyme

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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