Metallo \u3b2 lactamase: reactivity and site directed evolution pathways addressed by computational approaches

The indiscriminate prescription of antibiotics by physicians, along with their incorrect use [1] 1 has increased the exposition of bacteria to antibiotics, and thus has created a favorable environment for the Darwinian evolution of resistant strains [2]. Further increase of drug resistance is caused by the unnecessary massive use of antibiotics (70% of the total market is in the US!) to animals crammed into the unhygienic crowded quarters of factories [3, 4]. Diseases like tubercolosis, gonorrhea, malaria, and childhood ear infections, are increasingly becoming hard to treat with antibiotic drugs, posing serious concern in the human public health [5, 6]. The problem is even more serious if one considers that already in 70\u2019s and 80\u2019s that modification of the chemical structure of the already known antibiotics turned out to be exhausted and, at the same time, pharmacological companies decided not design of totally new antibiotics [2]

Metallo-β-lactamase-like enzymes from non-pathogenic organisms: an illustration of functional promiscuity

The spread of antibiotic resistance is one major global problem for healthcare systems. One of the most relevant mechanisms of resistance involves the expression, by bacteria, of enzymes able to degrade the antibiotic molecules. This thesis is focused on the study of a particular class of antibiotic-degrading enzymes, the metallo-β-lactamases (MBLs). MBLs are a family of Zn(II)-dependent enzymes that inactivate most of the commonly used β-lactam antibiotics. In Chapter 1 a detailed review of the properties of these enzymes is presented. Novel MBLs are continuously discovered and numerous variants of known MBLs emerge, largely due to the introduction and frequent misuse of novel antibiotics. It has also become apparent that MBLs are present in microorganisms that are not pathogenic and inhabit environments that are not likely to be subjected to significant evolutionary pressures (such as the sharp increase in antibiotics). MBLs from such environmental microorganisms may thus pose a future risk for health care, but they may also provide clues about essential factors that enable such enzymes to inactivate antibiotics. In Chapter 2 the discovery of two novel putative MBLs from the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans is described. In adherence with common practice these two enzymes were named Maynooth IMipenemase-1 (MIM-1) and Maynooth IMipenemase-2 (MIM-2), respectively. In Chapter 3 the biochemical properties of MIM-1 and MIM-2 are discussed and compared to those of known MBLs. From the pH dependence of their catalytic parameters it is evident that both enzymes differ with respect to their mechanisms, with MIM-1 preferring alkaline and MIM-2 acidic conditions. Both enzymes require Zn(II) but activity can also be reconstituted with other metal ions, including Co(II), Mn(II), Cu(II) and Ca(II). Importantly, the substrate preference of MIM-1 and MIM-2 appears to be influenced by their metal ion composition, which may be a relevant factor in determining their precise biological function. However, with respect to their catalytic efficiency towards degrading β-lactam antibiotics MIM-1 and MIM-2 are comparable to MBLs from known pathogenic bacteria such as Klebsiella pneumonia or Pseudomonas aeruginosa. They are also inhibited by the non-clinical compound D-captopril in a manner characteristic for MBLs. Thus, even though MIM-1 and MIM-2 are currently not associated with antibiotic resistance these enzymes, should they ever enter the human population, certainly could pose a future threat to health care. Since neither N. pentaromativorans nor S. agarivorans are human pathogens, the precise biological role(s) of MIM-1 and MIM-2 remains to be established. In Chapter 4 the possibility of an alternative function of MIM-1 and MIM-2 will be addressed. Although both protein sequence comparisons and homology modelling indicate that these proteins are related to well-known MBLs such as AIM-1, the sequence analysis also indicates that MIM-1 and MIM-2 share similarities with N-acyl homoserine lactonases (AHLases) and glyoxalase II (GLX-II). Steady-state kinetic assays using a series of lactone substrates confirm that MIM-1 and MIM-2 are indeed efficient lactonases, with catalytic efficiencies resembling those of well-known AHLases. Interestingly, unlike their MBL activity the AHLase activity of MIM-1 and MIM-2 is not dependent on the metal ion composition with Zn(II), Co(II), Cu(II), Mn(II) and Ca(II) all being able to reconstitute catalytic activity (with Co(II) being the most efficient). However, these enzymes do not turn over S-lactoylglutathione, a substrate characteristic for GLX-II activity. Since lactonase activity is linked to the process of quorum sensing the bifunctional activity of “non-pathogenic” MBLs such as MIM-1 and MIM-2 may provide insight into one possible evolutionary pathway for the emergence of antibiotic resistance. In the preceding chapters MIM-1 and MIM-2 were introduced as novel MBL-like enzymes that may provide essential clues about the functional promiscuity that may be inherent to the family of β-lactam-hydrolysing enzymes. It is thus essential to probe the catalytic mechanism of these enzymes in detail to gain insight into essential factors that control their reactivity. In Chapter 5 a series of physico-chemical experiments are described (including rapid kinetics and spectroscopic techniques) that provide insight into the active site structure and the mechanism of substrate turnover. In brief, while MIM-1 and MIM-2 employ a strategy that is similar to that of other MBLs by using a metal ion-activated hydroxide moiety to initiate the hydrolysis of β-lactam substrates (such as penicillin, cephalothin or imipenem) no reaction intermediate is observed. Such an intermediate is present in many MBL-characterised reactions and indicates that MIM-1 and MIM-2 may use a different mechanistic strategy, one where the identity of the rate-limiting step is different from that of many (but not all) MBLs. The characterisation of two novel members of the continuously growing family of MBLs has provided detailed insight into the structure and function of an antibiotic resistance mechanism that is not limited to pathogenic microorganisms. The similarity of the physico-chemical properties between MBLs from pathogenic and non-pathogenic sources may provide clues about evolutionary relationships that may underlie the rapid emergence and spread of antibiotic resistance, but it may also assist in the development of urgently needed potent and clinically useful inhibitors for this group of enzymes. In Chapter 6 some concluding remarks allude to future directions in this area of research, including a brief description of the crystal structures of both MIM-1 and MIM-2, which have been solved by another member of our team as this thesis was being completed. The hope remains that research like the one presented here will, in time, lead to a powerful strategy to combat the rise of antibiotic resistance and the grave dangers this brings to global human health