scholarly journals Biochemical and structural explorations of α-hydroxyacid oxidases reveal a four-electron oxidative decarboxylation reaction

2019 ◽  
Vol 75 (8) ◽  
pp. 733-742 ◽  
Author(s):  
Hsien-Wei Yeh ◽  
Kuan-Hung Lin ◽  
Syue-Yi Lyu ◽  
Yi-Shan Li ◽  
Chun-Man Huang ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Active Site ◽  
Oxidation Reaction ◽  
Isotope Labeling ◽  
Molecular Level ◽  
Flavin Mononucleotide ◽  
Oxidative Decarboxylation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Decarboxylation Reaction

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.

scholarly journals The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level

2019 ◽  
Vol 75 (10) ◽  
pp. 918-929
Author(s):  
Syue-Yi Lyu ◽  
Kuan-Hung Lin ◽  
Hsien-Wei Yeh ◽  
Yi-Shan Li ◽  
Chun-Man Huang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
High Resolution ◽  
Active Site ◽  
Oxidation Reaction ◽  
Substrate Oxidation ◽  
Flavin Mononucleotide ◽  
Oxidative Decarboxylation ◽  
Dead End ◽  
Decarboxylation Reaction ◽  
Free Hydrogen

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C′α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

scholarly journals Binding of Phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography

2001 ◽  
Vol 10 (8) ◽  
pp. 1669-1676 ◽  
Author(s):  
Demetres D. Leonidas ◽  
Gayatri B. Chavali ◽  
Anwar M. Jardine ◽  
Songlin Li ◽  
Robert Shapiro ◽  
...  
Keyword(s):  
Active Site ◽  
X Ray ◽  
X Ray Crystallography

An Oxidative Decarboxylation of 7-Oxopodocarpic Acid Derivatives

1990 ◽  
Vol 43 (5) ◽  
pp. 883 ◽  
Author(s):  
RC Cambie ◽  
HM Craw ◽  
CEF Rickard ◽  
JD Robertson ◽  
PS Rutledge ◽  
...  
Keyword(s):  
Optically Active ◽  
Oxidative Decarboxylation ◽  
X Ray ◽  
X Ray Crystallography

Treatment of the 7-oxopodocarpic acid derivatives (1) and (3) with 2,3-dichloro-5,6-dicyanobenzoquinone affords the conjugated dienones (5) and (6) directly. The latter compounds have potential as optically active relays for the synthesis of 3-oxygenated diterpenoids . The stereochemistry of an epoxide (7) formed from the dienone (5) has been confirmed by X-ray crystallography.

scholarly journals Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1211-C1211
Author(s):  
Joseph Ng ◽  
Ronny Hughes ◽  
Michelle Morris ◽  
Leighton Coates ◽  
Matthew Blakeley ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Crystallographic Structures ◽  
Hydrolysis Of ◽  
Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

scholarly journals Probing the active site of the sugar isomerase domain from E. coli arabinose-5-phosphate isomerase via X-ray crystallography

2010 ◽  
Vol 19 (12) ◽  
pp. 2430-2439 ◽  
Author(s):  
Louise J. Gourlay ◽  
Silvia Sommaruga ◽  
Marco Nardini ◽  
Paola Sperandeo ◽  
Gianni Dehò ◽  
...  
Keyword(s):  
Active Site ◽  
X Ray ◽  
E Coli ◽  
X Ray Crystallography

Utility of high resolution accurate mass spectrometry (HRMS) in the mass isotopomer distribution analysis (MIDA) of CSF proteins modified by stable isotope labeling in mammals (SILAM) methodology applied to neurodegenerative diseases

2017 ◽  
Vol 9 (23) ◽  
pp. 3477-3484
Author(s):  
Joseph L. Cantone ◽  
Craig Polson ◽  
Cong Wei ◽  
Valerie Guss ◽  
Michael K. Ahlijanian ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Isotope Labeling ◽  
Molecular Level ◽  
Disease Diagnosis ◽  
Stable Isotope Labeling ◽  
Distribution Analysis ◽  
Mass Isotopomer Distribution Analysis ◽  
Csf Proteins ◽  
Accurate Mass Spectrometry

Stable isotope labeling of proteins affords indicators at the molecular level, specifically biomarkers, which may providein vivodata on disease diagnosis, progression, and treatment.

scholarly journals Mechanism-based inactivator of isocitrate lyases 1 and 2 fromMycobacterium tuberculosis

2017 ◽  
Vol 114 (29) ◽  
pp. 7617-7622 ◽  
Author(s):  
Truc V. Pham ◽  
Andrew S. Murkin ◽  
Margaret M. Moynihan ◽  
Lawrence Harris ◽  
Peter C. Tyler ◽  
...  
Keyword(s):  
Active Site ◽  
High Efficiency ◽  
Isocitrate Lyase ◽  
Partition Ratio ◽  
Kinetic Properties ◽  
Ionization Mass ◽  
X Ray ◽  
X Ray Crystallography ◽  
Covalent Adduct ◽  
Reversible Covalent

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway forMycobacterium tuberculosis(Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator ofMtbICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191. 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalentS-homopyruvoyl adduct of the active-site Cys191.

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