Escherichia coli K-12 and some other strains have been reported to be capable of utilizing 3-(3-hydroxyphenyl)propionate (3HPP), one of the phenylpropanoids from lignin. Although other enzymes involved in 3HPP catabolism and their corresponding genes from its degraders have been identified, 3HPP 2-hydroxylase, catalyzing the first step of its catabolism, has yet to be functionally identified at biochemical and genetic levels. In this study, we investigated the function and characteristics of MhpA from E. coli strain K-12 (MhpA_K-12). Gene deletion and complementation showed that mhpA was vital for its growth on 3HPP, but the mhpA deletion strain was still able to grow on 3-(2,3-dihydroxyphenyl)propionate (DHPP), the hydroxylation product transformed from 3HPP by MhpA_K-12. MhpA_K-12 was overexpressed and purified, and it was likely a polymer and tightly bound with an approximately equal number of moles of FAD. Using NADH or NADPH as a cofactor, purified MhpA_K-12 catalyzed the conversion of 3HPP to DHPP at a similar efficiency. The conversion from 3HPP to DHPP by purified MhpA_K-12 was confirmed using high-performance liquid chromatography and liquid chromatography-mass spectrometry. Bioinformatics analysis indicated that MhpA_K-12 and its putative homologues belonged to taxa that were phylogenetically distant from functionally identified FAD-containing monooxygenases (hydroxylases). Interestingly, MhpA_K-12 has approximately an extra 150 residues at its C terminus in comparison to its close homologues, but its truncated versions MhpA_K-12⁴⁰⁰ and MhpA_K-12⁴⁸⁰ (with 154 and 74 residues deleted from the C terminus, respectively) both lost their activities. Thus, MhpA_K-12 has been confirmed to be a 3HPP 2-hydroxylase catalyzing the conversion of 3HPP to DHPP, the initial reaction of 3HPP degradation.

IMPORTANCE Phenylpropionate and its hydroxylated derivatives resulted from lignin degradation ubiquitously exist on the Earth. A number of bacterial strains have the ability to grow on 3HPP, one of the above derivatives. The hydroxylation was thought to be the initial and vital step for its aerobic catabolism via the meta pathway. The significance of our research is the functional identification and characterization of the purified 3HPP 2-hydroxylase MhpA from Escherichia coli K-12 at biochemical and genetic levels, since this enzyme has not previously been expressed from its encoding gene, purified, and characterized in any bacteria. It will not only fill a gap in our understanding of 3HPP 2-hydroxylase and its corresponding gene for the critical step in microbial 3HPP catabolism but also provide another example of the diversity of microbial degradation of plant-derived phenylpropionate and its hydroxylated derivatives.