Functional Metagenomics Reveals a New Catalytic Domain, the Metallo-β-Lactamase Superfamily Domain, Associated with Phytase Activity

Abstract

Inositol-6-phosphate, also known as phytic acid, is a phosphorus source that plays several important roles in the phosphorus cycle and in cell metabolism. The known characterized enzymes responsible for its degradation, the phytases, are mostly derived from cultured individual microorganisms. The catalytic signatures of phytases are restricted to the molecular domains of four protein superfamilies: histidine phosphatases, protein tyrosine phosphatases, the purple acid phosphatases and the β-propeller phosphatases. During function-based screening of previously generated forest soil metagenomic libraries for Escherichia coli clones conferring phytase activity, two positive clones harboring the plasmids pLP05 and pLP12 were detected. Analysis of the insert sequences revealed the absence of classic phosphatase/phytase signatures of the proteins deduced from the putative genes, but the genes mblp01 (pLP05) and mblp02 (pLP12) encoded putative metallo-β-lactamases (MBLs). Several MBL representatives are promiscuous proteins with phosphoesterase activity, but phytase activity was previously not reported. Both mblp01 and mblp02 were subcloned, expressed, and analyzed. Mblp01 and Mblp02 are members of the lactamase B2 family. Protein modeling showed that the closest structural homologue of both proteins was ZipD of E. coli. Mblp01 and Mblp02 showed activity toward the majority of the tested phosphorylated substrates, including phytate. The maximal enzyme activities were recorded for Mblp01 at 50°C under acidic conditions and for Mblp02 at 35°C and a neutral pH. In the presence of Cu2+ or SDS, the activities of Mblp01 and Mblp02 were strongly inhibited. Analyses of the minimal inhibitory concentrations of several β-lactam antibiotics revealed that recombinant E. coli cells carrying mblp01 or mblp02 showed reduced sensitivity toward β-lactam antibiotics. IMPORTANCE Phytic acid is a phosphorus storage molecule in many plant tissues, a source of phosphorus alternative to phosphate rocks, but it can also be a problematic antinutrient. In comparison to other phosphorus sources, phytic acid exhibits reduced bioavailability. Additionally, it influences functions of secondary messengers and acts as antioxidant in tumor growth prevention. The enzymatic capability to process phytate has been reported for a limited number of protein families. This might be due to the almost exclusive use of proteins derived from individual microorganisms to analyze phytase activity. With such a restriction, the study of the complexity and diversity of the phytases remains incomplete. By using metagenome-derived samples, this study demonstrates the existence of phytase activity in one of the most promiscuous superfamilies, the metallo-β-lactamases. Our results increase the general knowledge on phytase diversity in environmental samples and could provide new avenues for the study and engineering of new biocatalysts.

Structural and functional profile of phytases across the domains of life

“…HP2Ps and MINPPs), as well as previously unreported trends such as differences between plant PAPhys, and the identification of potential fungal BPPhys. Although only bacterial and eukaryotic phytases have been characterized so far, the recent report of metallo-β-lactamases with phytase activity may lead to the eventual discovery of archaeal phytases ( Castillo Villamizar et al, 2019a ).…”

Origin, Diversity, and Multiple Roles of Enzymes with Metallo-β-Lactamase Fold from Different Organisms

“…Recently, another atypical enzymatic activity of two MβL fold proteins has also been described from a functional metagenomic study of forest soil [ 34 ]. In this study, while the authors performed a function-based screening of libraries generated from the whole metagenomic sequence data of forest soil to identify positive phytase activity in E. coli clones, two clones were positive for this phytase activity.…”

“…As expected, the enzymatic characterisation revealed for both purified proteins an activity on the majority of tested phosphorylated substrates including phytate. Moreover, both purified enzymes were able to confer to recombinant E. coli strains less sensitivity to β-lactam antibiotics, suggestive of a β-lactamase activity, and qualified by the authors as promiscuous activity [ 34 ]. This promiscuous β-lactamase activity was also reported from the discovered and identified subclass B3 MβL protein, PNGM-1 from a conducted functional metagenomic analyses of deep-sea sediments predating the era of antibiotics [ 35 , 50 ].…”

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

“…Phytases in bacteria, fungi, plants and animals [ 6 , 7 ] are commonly classified by protein fold and catalytic mechanism. Four canonical phytases are commonly considered, the β-propeller phytases (BPPhy), Purple Acid Phytases (PAPhy), Protein Tyrosine Phosphatase-like Phytase (PTPLPs) (Cysteine phytases) and Histidine Acid Phytases (HAPhys) [ 8 ], but the classification has been extended by the characterization of soil metagenomes to include metallo-β-lactamase enzymes [ 9 , 10 ]. The histidine acid phytases are also comprised of a subclass, the Multiple Inositol Polyphosphate Phosphatases (MINPPs), which deviate from HAPhy sequence homology [ 11 ].…”