Characterization of a hyperthermophilic phosphatase from Archaeoglobus fulgidus and its application in in vitro synthetic enzymatic biosystem

Abstract

Background: Haloacid dehalogenase (HAD)-like hydrolases represent the largest superfamily of phosphatases, which release inorganic phosphate from phosphate containing compounds, such as sugar phosphates. The HAD-like phosphatases with highly substrate specificity, which perform irreversible dephosphorylation, are always integrated into in vitro synthetic enzymatic biosystems as the last enzymatic step for the cost-efficient production of biochemicals. Therefore, identification and characterization of substrate specificity of HAD-like phosphatases are important for exploring their application. Results: In this study, a hyperthermophilic HAD-like phosphatase from Archaeoglobus fulgidus (AfPase) was cloned, expressed, and characterized. AfPase was identified as a type I Mg 2+-dependent HAD-like phosphatase with high optimal temperature and thermostability. Among the tested phosphate containing compounds, AfPase exhibited the highest catalytic activity on p-nitrophenyl phosphate, followed by dihydroxyacetone phosphate (DHAP). On the basis of the high catalytic activity of AfPase to generate 1,3-dihydroxyacetone (DHA) from DHAP, an in vitro synthetic enzymatic biosystem containing this phosphatase and other five enzymes was constructed for the biosynthesis of DHA from inexpensive maltodextrin in one pot. About 14 mM (1.26 g/L) DHA was produced from 10 g/L maltodextrin. Conclusions: A hyperthermophilic HAD-like phosphatase from Archaeoglobus fulgidus was characterized carefully, and the success of an in vitro synthetic enzymatic biosystem containing this phosphatase provided a promising approach for DHA production from maltodextrin.

Structural Insights of a cis-Epoxysuccinate Hydrolase Facilitate the Development of Robust Biocatalysts for the Production of l-(+)-Tartrate

“…Moreover, it was verified through 18 O experiments , that the two-step reaction mechanism of CESHs bears a close resemblance to the catalytic process observed in l -DEXs (Scheme ). − Through site-directed mutagenesis, certain crucial catalytic residues have been identified, such as the Nu–His–Ac catalytic triad (D18–H190–D193 in Ro CESH, D48–H221–D224 in Ko CESH), where Nu is a nucleophilic aspartate residue and Ac is an acidic amino acid residue, facilitating the reception and release of protons to drive some specific chemical transformations. ,, Interestingly, the classic epoxide hydrolases from the α/β-hydrolase fold superfamily have the same “Asp–His–Asp” residues for their catalytic triad, but they have no sequence similarity with CESHs. , Their catalytic mechanism involves two additional tyrosine residues acting as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the nucleophile aspartate residue . Considering that both Ro CESH and Ko CESH contain three tyrosine residues around their active sites, it is possible that the same mechanism is also involved in the catalysis of CESHs.…”

Artificial ATP-Free in Vitro Synthetic Enzymatic Biosystems Facilitate Aldolase-Mediated C–C Bond Formation for Biomanufacturing

“…31 All four enzymes were overexpressed in E. coli and purified by Ni-NTA resin (Figure S2A in the Supporting Information). Since most phosphatases show promiscuous catalytic activity, 32 the substrate specificities of these four phosphatases on phosphate-containing substrates and intermediates included in this in vitro biosystem were determined (Table S1 in the Supporting Information). BsPase and BtPase showed comparable activity on DR5P with the intermediate products DHAP and FBP, indicating that these two phosphatases were not suitable for DR synthesis.…”