Crystal structure of the flavin reductase of Acinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPAH) and identification of amino acid residues underlying its regulation by aromatic ligands

The first step in the degradation of p-hydroxyphenylacetic acid (HPA) is catalyzed by the two-component enzyme p-hydroxyphenylacetate 3-hydroxylase (HPAH). The two components of Acinetobacter baumannii HPAH are known as C₁ and C₂, respectively. C₁ is a flavin reductase that uses NADH to generate reduced flavin mononucleotide (FMNH⁻), which is used by C₂ in the hydroxylation of HPA. Interestingly, although HPA is not directly involved in the reaction catalyzed by C₁, the presence of HPA dramatically increases the FMN reduction rate. Amino acid sequence analysis revealed that C₁ contains two domains: an N-terminal flavin reductase domain, and a C-terminal MarR domain. Although MarR proteins typically function as transcription regulators, the MarR domain of C₁ was found to play an auto-inhibitory role. Here, we report a crystal structure of C₁ and small-angle X-ray scattering (SAXS) studies that revealed that C₁ undergoes a substantial conformational change in the presence of HPA, concomitant with the increase in the rate of flavin reduction. Amino acid residues that are important for HPA binding and regulation of C₁ activity were identified by site-directed mutagenesis. Amino acid sequence similarity analysis revealed several as yet uncharacterized flavin reductases with N- or C-terminal fusions.