scholarly journals Structure of Allium lachrymatory factor synthase elucidates catalysis on sulfenic acid substrate

2017 ◽  
Author(s):  
Takatoshi Arakawa ◽  
Yuta Sato ◽  
Jumpei Takabe ◽  
Noriya Masamura ◽  
Masahiro Kato ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Sigmatropic Rearrangement ◽  
Natural Production ◽  
Lachrymatory Factor ◽  
Biological Decomposition ◽  
Key Residues ◽  
Lachrymatory Factor Synthase

AbstractNatural lachrymatory effects are invoked by small volatile S-oxide compounds. They are produced through alkene sulfenic acids by the action of lachrymatory factor synthase (LFS). Here we present the crystal structures of onion LFS (AcLFS) revealed in solute-free and two solute-stabilized forms. Each structure adopts a single seven-stranded helix-grip fold possessing an internal pocket. Mutagenesis analysis localized the active site to a layer near the bottom of the pocket, which is adjacent to the deduced key residues Arg71, Glu88, and Tyr114. Solute molecules visible on the active site have suggested that AcLFS accepts various small alcohol compounds as well as its natural substrate, and they inhibit this substrate according to their chemistry. Structural homologs have been found in the SRPBCC superfamily, and comparison of the active sites has demonstrated that the electrostatic potential unique to AcLFS could work in capturing the substrate in its specific state. Finally, we propose a rational catalytic mechanism based on intramolecular proton shuttling in which the microenvironment of AcLFS can bypass the canonical [1,4]-sigmatropic rearrangement principle established by microwave studies. Beyond revealing how AcLFS generates the lachrymatory compound, this study provides insights into the molecular machinery dealing with highly labile organosulfur species.Significance statementCrushing of onion liberates a volatile compound, syn-propanethial S-oxide (PTSO), which causes lachrymatory effect on humans. We present the crystal structures of onion LFS (AcLFS), the enzyme responsible for natural production of PTSO. AcLFS features a barrel-like fold, and mutagenic and inhibitory analyses revealed that the key residues are present in the central pocket, harboring highly concentrated aromatic residues plus a dyad motif. The architecture of AcLFS is widespread among proteins with various biological functions, such as abscisic acid receptors and polyketide cyclases, and comparisons with these homologs indicate that unique steric and electronic properties maintain the pocket as a reaction compartment. We propose the molecular mechanism behind PTSO generation and shed light on biological decomposition of short-lived sulfur species.

scholarly journals Short hydrogen bonds in the catalytic mechanism of serine proteases

2008 ◽  
Vol 73 (4) ◽  
pp. 393-403 ◽  
Author(s):  
Vladimir Leskovac ◽  
Svetlana Trivic ◽  
Draginja Pericin ◽  
Mira Popovic ◽  
Julijan Kandrac
Keyword(s):  
Hydrogen Bond ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Serine Proteases ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Data Bank ◽  
Ground State Structure ◽  
Low Barrier Hydrogen Bonds ◽  
Short Hydrogen Bonds

The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78-1.28 ? revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 ? long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short "low-barrier" hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the "low-barrier hydrogen bond" hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 ? long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the "low-barrier hydrogen bond" hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

Investigation of the Catalytic Properties of the Dysthrombin, Thrombin Quick

1979 ◽  
Author(s):  
R.A. Henriksen ◽  
W.G. Owen ◽  
M.E. Nesheim ◽  
K.G. Mann
Keyword(s):  
Active Site ◽  
Mayo Clinic ◽  
Active Sites ◽  
Fluorescence Enhancement ◽  
Catalytic Mechanism ◽  
Antithrombin Iii ◽  
Catalytic Properties ◽  
Inhibitor Binding ◽  
Equivalent Number ◽  
Aggregation Of Platelets

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and Ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of normal thrombin (T) The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. When incubated with antithrombin III, both T and TQ form inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinguished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 x 10-5M and kcat - 6.93 compared to Km = 4.0 × 10-5M and kcat = 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for tydrolysis of the active site titrant 4-methyumbel1 i feryl-p-guanidlnobenzoate.

scholarly journals The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

2001 ◽  
Vol 276 (15) ◽  
pp. 11698-11704 ◽  
Author(s):  
Pär L. Pettersson ◽  
Bengt Mannervik
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Ph Dependence ◽  
Enzymatic Catalysis ◽  
Catalytic Efficiency ◽  
Protonated Form ◽  
Hydroxyl Group ◽  
Isomerization Reaction

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Δ5-androstene-3,17-dione (AD) into Δ4-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect.S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr9into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pKavalue of the enzyme-bound glutathione thiol. Thus, Tyr9promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr9residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3β-hydroxysteroid dehydrogenase.

Photochemical mapping of the active site of myosin

1992 ◽  
Vol 336 (1276) ◽  
pp. 55-61 ◽  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Cross Linking ◽  
Detailed Structure ◽  
Atp Binding Site ◽  
Adenine Ring ◽  
Skeletal Myosin ◽  
Key Residues ◽  
Specific Labelling

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the γ-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.

scholarly journals Atomic-Level Investigation of Reactant Recognition Mechanism and Thermodynamic Property in Glucosamine 6-Phosphate Deaminase Catalysis

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiao Zhang ◽  
Xiaoyuan Liu ◽  
Zhiyang Zhang ◽  
Yuan Zhao ◽  
Chaojie Wang
Keyword(s):  
Hydrogen Bond ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Polar Solvent ◽  
Enzymatic Reaction ◽  
Alkaline Condition ◽  
Recognition Mechanism ◽  
Hydrogen Bond Interactions ◽  
Dynamics Simulations ◽  
Key Residues

Glucosamine 6-phosphate deaminase (NagB) influences the direction of N-acetylglucosamine (GlcNAc) metabolism, facilitating the conversion of D-glucosamine 6-phosphate (GlcN6P) to D-fructose 6-phosphate (Fru6P) with the release of ammonia. Here, extensive molecular dynamics simulations combined with various techniques were performed to study the recognition and delivery process of GlcN6P by SmuNagB, due to its guidance of subsequent enzymatic reaction. The key residues Lys194, His130, Arg127, Thr38, and Ser37 stabilize GlcN6P in the active site by hydrogen bond interactions, therein electrostatic and polar solvent effects provide the primary traction. Four delivery channels were identified, with GlcN6P most likely to enter the active site of NagB through a “door” comprising residues 6–10, 122–136, and 222–233. The corresponding mechanism and thermodynamic properties were investigated. An exothermic recognition and delivery process were detected, accompanied by the flipping of GlcN6P and changes in key direct and indirect hydrogen bond interactions, which provide the driving force for the chemical reaction to occur. Furthermore, “the lid motif” was identified that remain open in alkaline condition with different extent of opening at each stage of transfer that induced GlcN6P to move the active site of NagB. The work will assist in the elucidation of the catalytic mechanism of action of NagB, allowing inhibitors to be designed with superior dynamic behavior.

Investigation of the Catalytic Properties of the Dysthrombin, Thrombin Quick

1979 ◽  
Author(s):  
R Henriksen ◽  
W Owen ◽  
M Nesheim ◽  
K Mann
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Fluorescence Enhancement ◽  
Catalytic Mechanism ◽  
Antithrombin Iii ◽  
Catalytic Properties ◽  
Inhibitor Binding ◽  
Equivalent Number ◽  
Hydrolysis Of ◽  
Aggregation Of Platelets

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of nornar thrombin (T). The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. when incubated with antithrombin III, both T ad TQ fom inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinquished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 × 10-5M and kcat= 6.93 compared to Km = 4.0 × 10-5M and kcat= 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for hydrolysis of the active site titrant 4-methykl-umbelliferyl-p-guanidinobenzoate

Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

2019 ◽  
Vol 75 (12) ◽  
pp. 1138-1147
Author(s):  
Hsiao-Chuan Huang ◽  
Liu-Hong Qi ◽  
Yo-Chia Chen ◽  
Li-Chu Tsai
Keyword(s):  
Enzymatic Hydrolysis ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Sites ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Sites ◽  
Key Residues ◽  
Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

Structure of Hydrogenase in Biohydrogen Production Anaerobic Bacteria

2011 ◽  
pp. 251-258 ◽  
Author(s):  
Ming Du ◽  
Lu Zhang
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Anaerobic Bacteria ◽  
Research Direction ◽  
Biohydrogen Production ◽  
Future Research ◽  
Hydrogen Energy ◽  
Industrialization Process ◽  
Catalytic Mechanisms

Hydrogenase plays an important role in the process of biohydrogen production. Hydrogenases have very unique active sites and are classified into three groups according to the metal composition of the active sites: the [Ni-Fe] hydrogenase, [Fe-Fe] hydrogenase, and [Fe-only] hydrogenase. In this paper, the crystal structures and active sites of three kinds of hydrogenases are examined and compared. These enzymes have an unusual structural feature in common. Their similar active site indicates that the catalytic mechanism of hydrogen activation is probably similar. The understanding of the catalytic mechanisms for the three kinds of hydrogenases may help achieve the industrialization process of hydrogen energy production. Moreover, the future research direction about the hydrogenases from auto-aggregative bacteria and the chemical mimic of hydrogenases structure is discussed.

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