Biochemical investigations of polyphenol degradation enzymes in the phototrophic bacterium Rubrivivax gelatinosus.

Phloroglucinol (1,3,5-trihydroxybenzene) is an important intermediate in the degradation of flavonoids and tannins by anaerobic bacteria. Recent studies have shed light on the enzymatic mechanism of phloroglucinol degradation in butyrate-forming anaerobic bacteria, including environmental and intestinal bacteria such as Clostridium and Flavonifractor sp. Phloroglucinol degradation gene clusters have also been identified in other metabolically diverse bacteria, although the polyphenol metabolism of these microorganisms remain largely unexplored. Here, we describe biochemical studies of polyphenol degradation enzymes found in the purple non-sulfur bacterium Rubrivivax gelatinosus IL144, an anaerobic photoheterotroph reported to utilize diverse organic compounds as carbon sources for growth. In addition to the phloroglucinol reductase and dihydrophloroglucinol cyclohydrolase that catalyze phloroglucinol degradation, we characterize a Mn2+-dependent phloretin hydrolase that catalyzes the cleavage of phloretin into phloroglucinol and phloretic acid. We also report a Mn2+-dependent decarboxylase (DeC) that catalyzes the reversible decarboxylation of 2,4,6-trihydroxybenzoate to form phloroglucinol. A bioinformatics search led to the identification of DeC homologs in diverse soil and gut bacteria, and biochemical studies of a DeC homolog from the human gut bacterium Flavonifractor plautii demonstrated that it is also a 2,4,6-trihydroxybenzoate decarboxylase. Our study expands the range of enzymatic mechanisms for phloroglucinol formation, and provides further biochemical insight into polyphenol metabolism in the anaerobic biosphere.

Discovery and structural analysis of a phloretin hydrolase from the opportunistic human pathogen Mycobacterium abscessus.

The family of PhlG proteins catalyses the hydrolysis of carbon-carbon bonds and is widely distributed across diverse bacterial species. Two members of the PhlG family have been separately identified as 2,4-diacetylphloroglucinol (2,4-DAPG) hydrolase and phloretin hydrolase; however, the extent of functional divergence and catalytic substrates for most members of this family is still unknown. Here, using sequence similarity network and gene co-occurrence analysis, we categorized PhlG proteins into several subgroups and inferred that PhlG proteins from Mycobacterium abscessus (MaPhlG) are likely to be functionally equivalent to phloretin hydrolase. Indeed, we confirmed the hydrolytic activity of MaPhlG towards phloretin and its analog monoacetylphloroglucinol (MAPG), and the crystal structure of MaPhlG in complex with MAPG revealed the key residues involved in catalysis and substrate binding. Through mutagenesis and enzymatic assays, we demonstrated that H160, I162, A213 and Q266, which are substituted in 2,4-DAPG hydrolase, are essential for the activity towards phloretin. Based on the conservation of these residues, potential phloretin hydrolases were identified from Frankia, Colletotrichum tofieldiae and Magnaporthe grisea, which are rhizosphere inhabitants. These enzymes may be important for rhizosphere adaptation of the producing microbes by providing a carbon source through anaerobic degradation of flavonoids. Taken together, our results provided a framework for understanding the mechanism of functional divergence of PhlG proteins.