Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.
Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA ( Cg ) (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA (Cg) was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA(Cg). The pobA (Cg) is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.
Huang
Yan
Y
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Datun Road Jia-3#, Chaoyang District, Beijing, 100101, People's Republic of China.
Zhao
Ke-Xin
KX
Shen
Xi-Hui
XH
Jiang
Chen-Ying
CY
Liu
Shuang-Jiang
SJ
D001483:Base Sequence; D001665:Binding Sites; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D023061:Gene Order; D005798:Genes, Bacterial; D062385:Hydroxybenzoates / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009876:Operon; D010226:Parabens / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
C520290:3,4-dihydroxybenzoate; D062385:Hydroxybenzoates; D010226:Parabens; D009243:NAD; D009249:NADP; D006899:Mixed Function Oxygenases; C038193:4-hydroxybenzoic acid