Hydrolase metallo

Hydrolases that form an acyl-enzyme intermediate, such as some proteases and amidases, can be effectively used in this approach. On the other hand, this method is not applicable to metallo- and carboxyproteases. 

This chapter focuses on a novel antibiotic discovery paradigm. Metallo-hydrolases and Mur ligases are used to illustrate this approach. New methods to identify and prioritize targets, develop screens, and evaluate new inhibitors are discussed. New developments in enzyme-based assays, such as pathway assays, are also presented. This new approach is opening new venues for screening targets that are difficult to screen because substrates are not easily available. 

II. METALLO-HYDROLASES A FAMILY OF TARGETS FOR NOVEL ANTIBIOTIC DISCOVERY... 

A. Evaluation of Candidate Metallo-Hydrolases and Construction of Screening Strains... 

Table 1 Candidate Metallo-Hydrolases for New Antibiotic Discovery in Bacteria...

Figure 2 Antimicrobial compounds that inhibit metallo-hydrolases. NA, not applicable.

The greatly increased nucleophilicity of the catalytic serine distinguishes it from all other serine residues and makes it an ideal candidate for modification via activity-based probes [58]. Of the electrophilic probe types to profile serine hydrolases, the fluorophosphonate (FP)-based probes are the most extensively used and were first introduced by Cravatt and coworkers [38, 39]. FPs have been well-known inhibitors of serine hydrolases for over 80 years and were first applied as chemical weapons as potent acetylcholine esterase inhibitors. As FPs do not resemble a peptide or ester substrate, they are nonselective towards a particular serine hydrolase, thus allowing the entire family to be profiled. FPs also show minimal cross-reactivity with other classes of hydrolases such as cysteine-, metallo-, and aspartylhydrolases [59]. Furthermore, FP-based probes react only with the active serine hydrolase, and not the inactive zymogen, allowing these probes to interact only with functional species within the proteome [59]. Extensive use of this probe family has demonstrated their remarkable selectivity for serine hydrolases and resulted in the identification of over 100 distinct serine hydrolases... 

Since the release of HCN is a common defense mechanism for plants, the number of available HNLs is large. Depending on the plant family they are isolated from, they can have very different structures some resemble hydrolases or carbox-ypeptidase, while others evolved from oxidoreductases. Although many of the HNLs are not structurally related they all utilize acid-base catalysis. No co-factors need to be added to the reactions nor do any of the HNL metallo-enzymes require metal salts. A further advantage is that many different enzymes are available, R- or S-selective [10]. For virtually every application it is possible to find a stereoselective HNL (Table 5.1). In addition they tend to be stable and can be used in organic solvents or two-phase systems, in particular in emulsions. 

Enzymes that contain binuclear metal centers are also well suited to catalyze hydrolysis reactions, including a number of the reactions described above for the mononuclear metallohydrolases. Additionally, several of the examples that are discussed here belong to the enzyme superfamilies described above, specifically the amidohydrolase, zinc a,/3-hydrolase, and metallo-/31 superfamilies. The substrates for the binuclear metallohydrolases are also biologically diverse, including proteins, peptides, nucleotides, polyamines, and xenobiotics. The binuclear nature of these metal centers produces an active site with altered properties compared to the mononuclear counterparts, and therefore catalysis by these enzymes occurs with alternative reaction mechanisms. Readers are referred to the preceding sections for background information pertaining to enzymes that have already been discussed. 

Because over half of aU the commercially available antibiotics are 3-lactams, and in recent years bacterial resistance to these molecules has become so prevalent (to the point that some infections can no longer be treated), metaUo-jJ-lactamases are not only important from the organometallic standpoint but they are enzymes central to human health [229]. Most of the p-lactamases identified to date belong to the A, C and D serine hydrolase classes [230-233]. Class B enzymes, on the other hand, require divalent (Zn, Co, Cd or Mn) metal ions for catalytic activity [234, 235]. Because only 10% of the 200-odd P-lactamases identified so far are metaUoenzymes little attention has been given to these proteins as potential threats to antibiotic therapy [236,237]. However, metallo-p-lactamases are able to hydrolyze compoimds that are resistant to serine-p-lactamases and they show little or no susceptibility to traditional p-lactamase inhibitors [238, 239]. This has resulted in ineffective use of the most available antibiotics in bacteria capable of producing metaUo-P-lactamases [240, 241]. 

Classification of this enzyme as a serine, cysteine, aspartic, or metallo-dependent enzyme [50] is somewhat problematic because occasionally inhibitors from the same class led to contradictory results. The observed effects suggest, however, that both metal ions and sulfhydryl groups may play a major role in the hydrolytic mechanism. Therefore, most probably the enzyme is a metallocysteine hydrolase. 

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