Dual RNase and ß-lactamase Activity of a Single Enzyme Encoded in Archaea.

ß-lactam antibiotics have a well-known activity which disturbs the bacterial cell wall biosynthesis and may be cleaved by ß-lactamases. However, these drugs are not active on archaea microorganisms, which are naturally resistant because of the lack of ß-lactam target in their cell wall. Here, we describe that annotation of genes as ß-lactamases in Archaea on the basis of homologous genes is a remnant of identification of the original activities of this group of enzymes, which in fact have multiple functions, including nuclease, ribonuclease, ß-lactamase, or glyoxalase, which may specialized over time. We expressed class B ß-lactamase enzyme from Methanosarcina barkeri that digest penicillin G. Moreover, while weak glyoxalase activity was detected, a significant ribonuclease activity on bacterial and synthetic RNAs was demonstrated. The ß-lactamase activity was inhibited by ß-lactamase inhibitor (sulbactam), but its RNAse activity was not. This gene appears to have been transferred to the Flavobacteriaceae group especially the Elizabethkingia genus, in which the expressed gene shows a more specialized activity on thienamycin, but no glyoxalase activity. The expressed class C-like ß-lactamase gene, from Methanosarcina sp., also shows hydrolysis activity on nitrocefin and is more closely related to DD-peptidase enzymes. Our findings highlight the need to redefine the nomenclature of ß-lactamase enzymes and the specification of multipotent enzymes in different ways in Archaea and bacteria over time.

Promiscuous Enzyme Activity as a Driver of Allo and Iso Convergent Evolution, Lessons from the ß-Lactamases.

The probability of the evolution of a character depends on two factors: the probability of moving from one character state to another character state and the probability of the new character state fixation. The more the evolution of a character is probable, the more the convergent evolution will be witnessed, and consequently, convergent evolution could mean that the convergent character evolution results as a combination of these two factors. We investigated this phenomenon by studying the convergent evolution of biochemical functions. For the investigation we used the case of ß-lactamases. ß-lactamases hydrolyze ß-lactams, which are antimicrobials able to block the DD-peptidases involved in bacterial cell wall synthesis. ß-lactamase activity is present in two different superfamilies: the metallo-ß-lactamase and the serine ß-lactamase. The mechanism used to hydrolyze the ß-lactam is different for the two superfamilies. We named this kind of evolution an allo-convergent evolution. We further showed that the ß-lactamase activity evolved several times within each superfamily, a convergent evolution type that we named iso-convergent evolution. Both types of convergent evolution can be explained by the two evolutionary mechanisms discussed above. The probability of moving from one state to another is explained by the promiscuous ß-lactamase activity present in the ancestral sequences of each superfamily, while the probability of fixation is explained in part by positive selection, as the organisms having ß-lactamase activity allows them to resist organisms that secrete ß-lactams. Indeed, an organism that has a mutation that increases the ß-lactamase activity will be selected, as the organisms having this activity will have an advantage over the others.

The functional convergence of antibiotic resistance in ß-lactamases is not conferred by a simple convergent substitution of amino acid.

Bacterial resistance to antibiotics is a serious medical and public health concern worldwide. Such resistance is conferred by a variety of mechanisms, but the extensive variability in levels of resistance across bacteria is a common finding. Understanding the underlying evolutionary processes governing this functional variation in antibiotic resistance is important as it may allow the development of appropriate strategies to improve treatment options for bacterial infections. The main objective of this study was to examine the functional evolution of ß-lactamases, a common mechanism of enzymatic resistance that inactivates a widely used class of antibiotics. We first obtained ß-lactamase protein sequences and minimal inhibitory concentration (MIC), a measure of antibiotic function, from previously published literature. We then used a molecular phylogenetic framework to examine the evolution of ß-lactamase functional activity. We found that the functional activity of antibiotic resistance mediated by ß-lactamase has evolved in a convergent manner within molecular classes, but is not associated with any single amino acid substitution. This suggests that the dynamics of convergent evolution in this system can vary between the functional and molecular (sequence) levels. Such disassociation may hamper bioinformatic approaches to antibiotic resistance determination and underscore the need for (less efficient but more effective) activity assays as an essential step in evaluating resistance in a given case.

Human metallo-ß-lactamase enzymes degrade penicillin.

Nonribosomal peptides are assemblages, including antibiotics, of canonical amino acids and other molecules. ß-lactam antibiotics act on bacterial cell walls and can be cleaved by ß-lactamases. ß-lactamase activity in humans has been neglected, even though eighteen enzymes have already been annotated such in human genome. Their hydrolysis activities on antibiotics have not been previously investigated. Here, we report that human cells were able to digest penicillin and this activity was inhibited by ß-lactamase inhibitor, i.e. sulbactam. Penicillin degradation in human cells was microbiologically demonstrated on Pneumococcus. We expressed a MBLAC2 human ß-lactamase, known as an exosome biogenesis enzyme. It cleaved penicillin and was inhibited by sulbactam. Finally, ß-lactamases are widely distributed, archaic, and have wide spectrum, including digesting anticancer and ß-lactams, that can be then used as nutriments. The evidence of the other MBLAC2 role as a bona fide ß-lactamase allows for reassessment of ß-lactams and ß-lactamases role in humans.

An Integrative Database of ß-Lactamase Enzymes: Sequences, Structures, Functions, and Phylogenetic Trees.

ß-Lactamase enzymes have attracted substential medical attention from researchers and clinicians because of their clinical, ecological, and evolutionary interest. Here, we present a comprehensive online database of ß-lactamase enzymes. The current database is manually curated and incorporates the primary amino acid sequences, closest structural information in an external structure database (the Protein Data Bank [PDB]) and the functional profiles and phylogenetic trees of the four molecular classes (A, B, C, and D) of ß-lactamases. The functional profiles are presented according to the MICs and kinetic parameters that make them more useful for the investigators. Here, a total of 1,147 ß-lactam resistance genes are analyzed and described in the database. The database is implemented in MySQL and the related website is developed with Zend Framework 2 on an Apache server, supporting all major web browsers. Users can easily retrieve and visualize biologically important information using a set of efficient queries from a graphical interface. This database is freely accessible at http://ifr48.timone.univ-mrs.fr/beta-lactamase/public/.

Phylogenomic Analysis of ß-Lactamase in Archaea and Bacteria Enables the Identification of Putative New Members.

ß-lactamases are enzymes which are commonly produced by bacteria and which degrade the ß-lactam ring of ß-lactam antibiotics, namely penicillins, cephalosporins, carbapenems, and monobactams, and inactivate these antibiotics. We performed a rational and comprehensive investigation of ß-lactamases in different biological databases. In this study, we constructed hidden Markov model profiles as well as the ancestral sequence of four classes of ß-lactamases (A, B, C, and D), which were used to identify potential ß-lactamases from environmental metagenomic (1206), human microbiome metagenomic (6417), human microbiome reference genome (1310), and NCBI's nonredundant databases (44101). Our analysis revealed the existence of putative ß-lactamases in the metagenomic databases, which appeared to be similar to the four different molecular classes (A-D). This is the first report on the large-scale phylogenetic diversity of new members of ß-lactamases, and our results revealed that metagenomic database dark-matter contains ß-lactamase-like antibiotic resistance genes.