Observation of a Short, Strong Hydrogen Bond in the Active Site of Hydroxynitrile Lyase from Hevea brasiliensis Explains a Large pK Shift of the Catalytic Base Induced by the Reaction Intermediate

Stranzl, G.R.; Gruber, Karl; Steinkellner, Georg; Zangger, Klaus; Schwab, Helmut; Kratky, Christoph

doi:10.1074/jbc.m306814200

Cited by 37 publications

(49 citation statements)

References 57 publications

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“…Such a strong, short hydrogen bond has previously been both calculated (15) and experimentally monitored (29). In our models, it is formed only in those settings from which cyanohydrine is synthesized (QC2 and QC5 in Table 6), confirming its crucial role.…”

Section: Discussionsupporting

confidence: 52%

“…This means that upon binding of the cyanide compound, His 235 would turn into a powerful agent for the deprotonation of HCN. The pK a of His 235 is ϳ4 in the free enzyme (15,28,29), and indeed this residue appears deprotonated in the NAT structure. This indicates that the incoming ligand(s) severely affect the pK a of the catalytic residues and, together with the apparent high mobility of the protons in the active site observed from the QC results, supports the idea that the reaction mechanism functions via a protonation-deprotonation sequence (15).…”

Section: Discussionmentioning

confidence: 99%

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Atomic Resolution Crystal Structures and Quantum Chemistry Meet to Reveal Subtleties of Hydroxynitrile Lyase Catalysis

Schmidt

Gruber

Kratky

et al. 2008

Journal of Biological Chemistry

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Hydroxynitrile lyases are versatile enzymes that enantiospecifically cope with cyanohydrins, important intermediates in the production of various agrochemicals or pharmaceuticals. We determined four atomic resolution crystal structures of hydroxynitrile lyase from Hevea brasiliensis: one native and three complexes with acetone, isopropyl alcohol, and thiocyanate. We observed distinct distance changes among the active site residues related to proton shifts upon substrate binding. The combined use of crystallography and ab initio quantum chemical calculations allowed the determination of the protonation states in the enzyme active site. We show that His 235 of the catalytic triad must be protonated in order for catalysis to proceed, and we could reproduce the cyanohydrin synthesis in ab initio calculations. We also found evidence for the considerable pK a shifts that had been hypothesized earlier. We envision that this knowledge can be used to enhance the catalytic properties and the stability of the enzyme for industrial production of enantiomerically pure cyanohydrins.Hydroxynitrile lyases (HNLs), 2 EC 4.2.1.39, catalyze the cleavage of cyanohydrins into hydrocyanic acid (HCN) and the corresponding aldehyde or ketone (Fig. 1). In nature, the liberation of HCN serves as a defense mechanism against herbivores and microbial attack in a large variety of plants (1-3) and is initiated by a ␤-glycosidase-mediated degradation of cyanoglycosides, yielding a sugar and the ␣-hydroxynitrile (cyanohydrin). HNL is therefore one of the key targets for disruption of this pathway in order to prevent the HCN liberation and thus to detoxify food crops. In vitro, the (reverse) cyanohydrin synthesis reaction is favored (4), and HNLs have already been efficiently used for industrial syntheses of optically pure chiral cyanohydrins (4 -7), important intermediates for the production of a wide range of pharmaceuticals and agrochemicals (8). An overview of HNL function and application has been given by Johnson et al. (9) and references therein.Hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) is an FAD-independent HNL (2), an unglycosylated protein of the ␣/␤ hydrolase fold family with a molecular mass of 29.2 kDa that occurs as a homodimer in neutral aqueous solution (10). In vitro Hb-HNL accepts a variety of aliphatic, aromatic, and heterocyclic aldehydes or ketones for the (S)-specific synthesis of the corresponding ␣-cyanohydrins (4, 6, 11). Although the three-dimensional structures of the native enzyme (12, 13) and of a number of complexes (14 -16) have been known for some time, fine details of substrate interaction, such as the protonation state or subtle active site rearrangement, or structural data on a ternary complex with both substrates have so far been elusive. One problem was the limitation in crystallographic resolution for the complexes. Another problem was posed by the "stickiness" of the active site, which resulted in unwanted complexes at high resolution with histidine (used as a buffer in the first samples (12)) or...

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Section: Discussionsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Atomic Resolution Crystal Structures and Quantum Chemistry Meet to Reveal Subtleties of Hydroxynitrile Lyase Catalysis

Schmidt

Gruber

Kratky

et al. 2008

Journal of Biological Chemistry

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“…When these enzymes interact with their mechanism-based inhibitors, the imidazole ring in the side chain of the central histidine is protonated, and its hydrogen bond with the acidic residue is greatly strengthened, leading to a significant downfield shift of the corresponding proton signal into the 16 -19.5-ppm range (45)(46)(47). Based on these facts, we used NMR spectroscopy to detect the proton in the strong hydrogen bond between the side chains of Asp 201 and His 232 in the catalytic triad of 15 N-labeled MenH.…”

Section: Resultsmentioning

confidence: 99%

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

Sun

Yin

Feng

et al. 2014

Journal of Biological Chemistry

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“…Hydroxynitrile lyase:thiocyanate complex: Stranzl et al 74 have shown by NMR spectroscopy that the pK a value of His235 in hydroxynitrile lyase increases from 2.5 to $8 upon binding of thiocyanate. The current version of PROPKA 2.0 does not treat S atoms as an ionizable group.…”

Section: Charged Ligandsmentioning

confidence: 99%

Very fast prediction and rationalization of pK_a values for protein–ligand complexes

2008

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The PROPKA method for the prediction of the pKa values of ionizable residues in proteins is extended to include the effect of non‐proteinaceous ligands on protein pKa values as well as predict the change in pKa values of ionizable groups on the ligand itself. This new version of PROPKA (PROPKA 2.0) is, as much as possible, developed by adapting the empirical rules underlying PROPKA 1.0 to ligand functional groups. Thus, the speed of PROPKA is retained, so that the pKa values of all ionizable groups are computed in a matter of seconds for most proteins. This adaptation is validated by comparing PROPKA 2.0 predictions to experimental data for 26 protein–ligand complexes including trypsin, thrombin, three pepsins, HIV‐1 protease, chymotrypsin, xylanase, hydroxynitrile lyase, and dihydrofolate reductase. For trypsin and thrombin, large protonation state changes (|n| > 0.5) have been observed experimentally for 4 out of 14 ligand complexes. PROPKA 2.0 and Klebe's PEOE approach (Czodrowski P et al. J Mol Biol 2007;367:1347–1356) both identify three of the four large protonation state changes. The protonation state changes due to plasmepsin II, cathepsin D and endothiapepsin binding to pepstatin are predicted to within 0.4 proton units at pH 6.5 and 7.0, respectively. The PROPKA 2.0 results indicate that structural changes due to ligand binding contribute significantly to the proton uptake/release, as do residues far away from the binding site, primarily due to the change in the local environment of a particular residue and hence the change in the local hydrogen bonding network. Overall the results suggest that PROPKA 2.0 provides a good description of the protein–ligand interactions that have an important effect on the pKa values of titratable groups, thereby permitting fast and accurate determination of the protonation states of key residues and ligand functional groups within the binding or active site of a protein. Proteins 2008. © 2008 Wiley‐Liss, Inc.

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Observation of a Short, Strong Hydrogen Bond in the Active Site of Hydroxynitrile Lyase from Hevea brasiliensis Explains a Large pK Shift of the Catalytic Base Induced by the Reaction Intermediate

Cited by 37 publications

References 57 publications

Atomic Resolution Crystal Structures and Quantum Chemistry Meet to Reveal Subtleties of Hydroxynitrile Lyase Catalysis

Atomic Resolution Crystal Structures and Quantum Chemistry Meet to Reveal Subtleties of Hydroxynitrile Lyase Catalysis

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

Very fast prediction and rationalization of pK_a values for protein–ligand complexes

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Observation of a Short, Strong Hydrogen Bond in the Active Site of Hydroxynitrile Lyase from Hevea brasiliensis Explains a Large pK Shift of the Catalytic Base Induced by the Reaction Intermediate

Cited by 37 publications

References 57 publications

Atomic Resolution Crystal Structures and Quantum Chemistry Meet to Reveal Subtleties of Hydroxynitrile Lyase Catalysis

Atomic Resolution Crystal Structures and Quantum Chemistry Meet to Reveal Subtleties of Hydroxynitrile Lyase Catalysis

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

Very fast prediction and rationalization of pKa values for protein–ligand complexes

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Product

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Very fast prediction and rationalization of pK_a values for protein–ligand complexes