Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester.
L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K(d) 6.7 x 10(-7) M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavin-binding site of the enzyme, apo-L-aspartate oxidase has been reacted with N6-(6-carboxyhexyl)-FAD succinimidoester. The structural characterization of the resulting N6-(6-carbamoylxyhexyl)FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/monomer. Residue Lys38 was identified as the target of the covalent modification. N6-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.
Negri
A
A
Istituto di Fisioilogia Veterinaria e Biochimica, Università di Milano, Milan, Italy. armando.negri@unimi.it
Buckmann
A F
AF
Tedeschi
G
G
Stocker
A
A
Ceciliani
F
F
Treu
C
C
Ronchi
S
S
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D004952:Esters; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D029968:Escherichia coli Proteins; D004952:Esters; C107268:N(6)-(6-carboxyhexyl)FAD; D005182:Flavin-Adenine Dinucleotide; D000594:Amino Acid Oxidoreductases; C031955:L-aspartate oxidase, E coli