One origin for metallo-β-lactamase activity, or two? An investigation assessing a diverse set of reconstructed ancestral sequences based on a sample of phylogenetic trees

Abstract

Bacteria use metallo-β-lactamase enzymes to hydrolyse lactam rings found in many antibiotics, rendering them ineffective. Metallo-β-lactamase activity is thought to be polyphyletic, having arisen on more than one occasion within a single functionally diverse homologous superfamily. Since discovery of multiple origins of enzymatic activity conferring antibiotic resistance has broad implications for the continued clinical use of antibiotics, we test the hypothesis of polyphyly further; if lactamase function has arisen twice independently, the most recent common ancestor (MRCA) is not expected to possess lactam-hydrolysing activity. Two major problems present themselves. Firstly, even with a perfectly known phylogeny, ancestral sequence reconstruction is error prone. Secondly, the phylogeny is not known, and in fact reconstructing a single, unambiguous phylogeny for the superfamily has proven impossible. To obtain a more statistical view of the strength of evidence for or against MRCA lactamase function, we reconstructed a sample of 98 MRCAs of the metallo-β-lactamases, each based on a different tree in a bootstrap sample of reconstructed phylogenies. InterPro sequence signatures and homology modelling were then used to assess our sample of MRCAs for lactamase functionality. Only 5 % of these models conform to our criteria for metallo-β-lactamase functionality, suggesting that the ancestor was unlikely to have been a metallo-β-lactamase. On the other hand, given that ancestral proteins may have had metallo-β-lactamase functionality with variation in sequence and structural properties compared with extant enzymes, our criteria are conservative, estimating a lower bound of evidence for metallo-β-lactamase functionality but not an upper bound.

Fig. 1

Schematic diagram of the MRCA approach using a bootstrap sample. Additional sequences were aligned to the pre-existing FunTree alignment. This alignment was then used to build a maximum-likelihood tree, with 100 bootstrap replicates. The MRCA sequence was obtained from 98 of the trees in the bootstrap set. The 98 sequences were clustered at 60 % sequence identity. A representative from each cluster was submitted to the homology modelling server PHYRE2. Functional analysis was then carried out on each of these homology models compared to pre-constructed active site templates

Fig. 2

Rooted phylogenetic tree with percentage bootstrap support values. Tips are identified by UniProtKB accession numbers. Enzyme groups are colour coded by function as follows—red ribonucleases, cyan glyoxalase IIs, green A-type flavoproteins, pale pink subclass B2 metallo-β-lactamases, magenta B1 metallo-β-lactamases, orange B3 metallo-β-lactamases, black no function assigned. The phylogeny was visualised using Mesquite (Maddison and Maddison 2011)

Fig. 3

Representative sequences from each of the 11 clusters with weightings. Sequence alignment of each representative from each cluster, aligned with default settings in MAFFT. Each percentage value represents the weight of the cluster from the 44 MRCA sequences with IPR001018 signatures. Columns are coloured at a 70 % similarity threshold. The sequence alignment was visualised in BioEdit (Hall 1999)

Fig. 4

Homology model of an MRCA with possible metallo-β-lactamase functionality. The sequence representative number 51 passed both criteria for being most like a B3 metallo-β-lactamase. The PHYRE2 homology model (cartoon, rainbow) aligned with 1SML with an RMSD of 3.5 angstroms. A zoomed in image of each homology model’s predicted catalytic residues is shown. Homology model predicted residues ASP (green) aligned with catalytic ASP residues in the B3 template (grey). Homology model predicted residues TYR (orange) aligned with catalytic TYR residues in the B3 template (grey). Distances are shown in angstroms. Image was generated using Pymol (Schrodinger LLC 2010)