Structural Analysis of Flavinylation in Vanillyl-Alcohol Oxidase

Thr). In the mutant enzyme the covalent His-C8 alpha -flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K-d = 1.8 and 2.1 muM, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-1), K-m = 40 muM) than the wild-type enzyme. The crystal structures of both the hole and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-BI plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site. -->

Popis souboru:	application/octet-stream; application/pdf
ISSN:	0021-9258
DOI:	10.1074/jbc.m004753200
Přístupová URL adresa:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2da937452f10d116bf6805a7274ed2b6 https://doi.org/10.1074/jbc.m004753200
Rights:	OPEN
Přírůstkové číslo:	edsair.doi.dedup.....2da937452f10d116bf6805a7274ed2b6
Autor:	Fraaije, MW, van den Heuvel, RHH, van Berkel, WJH, Mattevi, A, Heuvel, Robert H.H. van den, Berkel, Willem J.H. van
Přispěvatelé:	University of Groningen, Groningen Biomolecular Sciences and Biotechnology, Biotechnology
Rok vydání:	2000
Předmět:	Models Molecular Vanillyl-alcohol oxidase Flavin Mononucleotide Protein Conformation Stereochemistry Molecular Conformation Biochemie Flavoprotein Crystallography X-Ray Biochemistry Protein Structure Secondary P-HYDROXYBENZOATE HYDROXYLASE Cofactor Apoenzymes Oxidoreductase PROTEIN MODELS BINDING Escherichia coli Life Science Point Mutation Histidine CRYSTAL-STRUCTURES L-aspartate oxidase Binding site Molecular Biology VLAG chemistry.chemical_classification Oxidase test Binding Sites REFINEMENT biology ACTIVE-SITE FAD Penicillium Active site Cell Biology Recombinant Proteins OXIDOREDUCTASE FAMILY Adenosine Diphosphate Alcohol Oxidoreductases FLAVOPROTEIN chemistry L-ASPARTATE OXIDASE Flavin-Adenine Dinucleotide Mutagenesis Site-Directed biology.protein
Zdroj:	Journal of Biological Chemistry, 275, 38654-38658 The Journal of Biological Chemistry, 275(49), 38654-38658. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC Journal of Biological Chemistry 275 (2000)
ISSN:	0021-9258
DOI:	10.1074/jbc.m004753200
Popis:	Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8 alpha -flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K-d = 1.8 and 2.1 muM, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-1), K-m = 40 muM) than the wild-type enzyme. The crystal structures of both the hole and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-BI plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.
Databáze:	OpenAIRE
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