PDBsum entry 6a1h

Flavoprotein

PDB id: 6a1h

Contents

Protein chain

332 a.a.

Ligands

9O9

Waters ×440

PDB id:

6a1h

Name:

Flavoprotein

Title:

Mandelate oxidase mutant-y128f with 5-deazariboflavin mononucleotide

Structure:

4-hydroxymandelate oxidase. Chain: a. Engineered: yes. Mutation: yes

Source:

Amycolatopsis orientalis. Nocardia orientalis. Organism_taxid: 31958. Gene: hmo. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.36Å R-factor: 0.169 R-free: 0.181

Authors:

T.L.Li,K.H.Lin

Key ref:

S.Y.Lyu et al. (2019). The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level. Acta Crystallogr D Struct Biol, 75, 918-929. PubMed id: 31588923 DOI: 10.1107/S2059798319011938

Date:

07-Jun-18 Release date: 19-Jun-19

Protein chain

O52792  (HMO_AMYOR) -  4-hydroxymandelate oxidase from Amycolatopsis orientalis

Seq: 357 a.a.

Struc: 332 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.1.3.46 - 4-hydroxymandelate oxidase.
• Reaction: (S)-4-hydroxymandelate + O2 = 4-hydroxyphenylglyoxylate + H2O2
• Cofactor: FMN

FMN (Bound ligand (Het Group name = 9O9) matches with 93.75% similarity)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1107/S2059798319011938

Acta Crystallogr D Struct Biol 75:918-929 (2019)

PubMed id: 31588923

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

S.Y.Lyu, K.H.Lin, H.W.Yeh, Y.S.Li, C.M.Huang, Y.L.Wang, H.W.Shih, N.S.Hsu, C.J.Wu, T.L.Li.

ABSTRACT

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 (FMN_ox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMN_ox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMN_red dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMN_red C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMN_ox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.