Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> |
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Authors: | Yan Huang Ke-xin Zhao Xi-Hui Shen Chen-Ying Jiang Shuang-Jiang Liu |
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Institution: | (1) 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 |
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Abstract: | 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 PobACg. 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. |
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Keywords: | 4-Hydroxybenzoate hydroxylase Corynebacterium glutamicum 4-Hydroxybenzoate Protochatechuate |
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