Question : What is CepA?

Answer : CepA is a chromosomal class A Beta-lactamase produced by the Enterobacteria Bacteroides fragilis with a wide-ranging substrate hydrolysis profile. CepA shows efficient hydrolysis of cephalosporins. It is inhibited by Clavulanic acid and tazobactam but not by aztreonam. But a-methoxyl cephamycins such as cefoxitin and the carbapenems have been highly active against Bacteroides species.

Question : How many natural variants of CepA are reported?

Answer : A total of 23 sequences were reported and two species of Bacteroides were included, 22 sequences isolated from Bacteroides fragilis and one isolated from Bacteroides cellulosilyticus.

Question : Why have we renamed the variants and how is it done?

Answer : To prepare a scientific basis for a new nomenclature system that can define all sub-classes of β-lactamases, since the present nomenclature system is simply based on the order in which they were found. The Renaming was done following a new nomenclature system in which CepA_AAA21532.1_Beta-lactamase was designated as cepA wild type and the rest of the variants from both the species previously mentioned were assigned a primary ID. Such a nomenclature ensured no overlapping among the variants when being addressed. The nomenclature syntax is: cepA_[Primary ID]_[accession_no.]|hlab. Note: The accession number is given as collected from each database.

Question : Where are the important loops and motifs of CepA located?

Answer : SVFK(70-73), SDN(130-132), KTG(234-236). The omega loop, although present in CepA has a different set of amino acid residues as compared to other prevalently studied Class A Beta-lactamases such as TEM, SHV, and CTX-M and is similar to PER-1. A single variable residue is observed among the common conserved regions at position 171. It is part of the omega loop, 14 of the 23 residues have a glutamate residue instead of lysine.

Question : How did we model the structures?

Answer : There is no crystal structure available on the CEPA enzyme obtained from Bacteroides fragilis. The structures were modeled based on homology modeling, the template chosen for all the variants was an apo crystal structure of Bacteroides cellulosilyticus, which showed the highest similarity percentage in all the cases.

Question : What is the significance of variant analysis of CepA?

Answer : CEPA shows a mutable frequency of 7.35% (20 out of 272 residues show a substitution mutation). A Mutational landscape was prepared with a target to identify mutational hotspots. The sequence of all 22 variants of CepA was compared against the Wild-type CepA_WT. The format followed a list of variants having the mutation and substitution mutation it underwent at a particular position.

Question : How did we choose the simulation candidates?

Answer : We have chosen primarily the candidates based on the following categories: The variants having the high number of mutations if any variants have mutations within or in the neighboring regions of the catalytic sites, and if any novel mutation was observed. Following these criteria, CepA simulation candidates included CepA_04, CepA_09, CepA_14, and CepA_12. Among a few unique substitution mutations observed in CepA sequence analysis, variant CepA_20 with a substitution mutation of H153N was also considered a simulation candidate.