MhpA Is a Hydroxylase Catalyzing the Initial Reaction of 3-(3-Hydroxyphenyl)Propionate Catabolism in Escherichia coli K-12

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, is yet to be functionally identified at biochemical and genetic levels. In this study, we investigated the function and characters of MhpAK-12 from strain K-12. Gene deletion and complementation showed that mhpA was vital for its growth on 3HPP, but the mhpA-deleted strain was still able to grow on 3-(2,3-dihydroxyphenyl)propionate (DHPP), the hydroxylation product transformed from 3HPP by MhpAK-12. MhpAK-12 was overexpressed and purified, and it was likely a polymer and tightly bound with approximately equal mole number of FAD. Using NADH or NADPH as cofactor, purified MhpAK-12 catalyzed the conversion of 3HPP to DHPP at a similar efficiency. The conversion from 3HPP to DHPP by purified MhpAK-12 was confirmed using high-performance liquid chromatography and liquid chromatography-mass spectrometry. Bioinformatics analysis indicated that MhpAK-12 and its putative homologues belonged to phylogenetically distant taxa comparing with functionally identified members from the FAD_binding_3 family. Interestingly, MhpAK-12 has extra approximately 150 residues at its C-terminus in comparison with its close homologues, but its truncated versions MhpAK-12400 and MhpAK-12480 (with 154 and 74 residues deletion from the C-terminus, respectively) both lost their activities. Thus MhpAK-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 MhpAK-12 from E. 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.