A promiscuous ancestral enzyme´s structure unveils protein variable regions of the highly diverse metallo-β-lactamase family

Abstract

The metallo-β-lactamase fold is an ancient protein structure present in numerous enzyme families responsible for diverse biological processes. The crystal structure of the hyperthermostable crenarchaeal enzyme Igni18 from Ignicoccus hospitalis was solved at 2.3 Å and could resemble a possible first archetype of a multifunctional metallo-β-lactamase. Ancestral enzymes at the evolutionary origin are believed to be promiscuous all-rounders. Consistently, Igni18´s activity can be cofactor-dependently directed from β-lactamase to lactonase, lipase, phosphodiesterase, phosphotriesterase or phospholipase. Its core-domain is highly conserved within metallo-β-lactamases from Bacteria, Archaea and Eukarya and gives insights into evolution and function of enzymes from this superfamily. Structural alignments with diverse metallo-β-lactamase-fold-containing enzymes allowed the identification of Protein Variable Regions accounting for modulation of activity, specificity and oligomerization patterns. Docking of different substrates within the active sites revealed the basis for the crucial cofactor dependency of this enzyme superfamily.

Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater

“…Structural model comparisons revealed that enzymes shared an average of 1.73 ± 0.43 angstrom (Å) rootmean squared deviation (RMSD) of atomic positions with the P. putida reference DH (Table S6). This is on par with RMSD values measured within other contaminant-degrading enzyme families sharing similar functions (Lakshminarasimhan et al, 2010;Perez-Garcia et al, 2021;Sidhu et al, 2019). We then compared our sequence phylogeny (Figure 2B) with a neighbor-joining tree generated from the RMSD matrix of all-vs-all structural alignments in our AlphaFold2 library (Figure 2C).…”

The metagenome‐derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET)

“…The lactonase activity of PET 40 was investigated by performing a phenol red‐assay according to Perez‐Garcia et al . [49]. As substrates, δ‐dodecalactone, γ‐dodecalactone, γ‐caprolactone and δ‐octalactone (Sigma‐Aldrich, Munich, Germany) were chosen, which release protons upon hydrolysis resulting in a pH shift.…”

“…To obtain insights into the potential binding modes of the diverse substrates in the active site of PET40, we performed molecular docking using a combination of AutoDock3 [46] as a docking engine and DrugScore 2018 [47,48] as an objective function as done previously [24, 49,50]. We focused on the ester substrates investigated experimentally in this study for the docking, that is mono(2-hydroxyethyl) terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), PET trimer (PET 3 ), methyl hexanoate (MH), polycaprolactone trimer (PCL 3 ), propane-1,2,3-triyl tributanoate (tributyrin, TBT), 4-nitrophenyl decanoate (pNP-C10), and 4-nitrophenyl dodecanoate (pNP-C12).…”

Multifunctional Biocatalysts for Organic Synthesis

“…For example, a generalist and multifunctional enzyme that combines three activities was identified by Chen et al while studying the evolution of a lower order algal lipoxygenase PhLOX (Scheme a) . This evolutionary hypothesis is also supported by ancestral sequence reconstruction, which curates an artificial sequence space from multiple homologues alignments and claim that the number of specialist enzymes correlate with evolutionary time. − This technique could offer a convenient method for creating new MFBs. Equally, MFBs that employ a single active site are common within biosynthesis including the well-known ATP citrate lyase reaction, as well as the remarkable 5-step macrofomate synthase reaction, and could be a source of inspiration. , In anisomycin biosynthesis the multifunctional dehydrogenase AniN is capable of both alcohol oxidation and imine reduction (Scheme b), which in biocatalysis is well explored as a valuable hydrogen borrowing cascade using separate biocatalysts. , More recently a single amine dehydrogenase, a so-called “alcohol aminase”, has been reported that catalyzes concurrent alcohol oxidation and reductive amination with ammonia .…”