Gianpiero Garau

Update of the Standard Numbering Scheme for Class B β-Lactamases

β-Lactamases represent the major cause of bacterial resistance against β-lactam antibiotics, and they have been divided into four classes (A to D) on the basis of their amino acid sequences (21). The class B enzymes have no sequence or structural similarity to the active-site serine enzymes of classes A, C, and D (6); require a bivalent metal ion (Zn2+) for activity; and constitute group 3 in the Bush-Jacoby-Medeiros functional classification (2). The identification of Zn-β-lactamase-producing pathogenic strains of Aeromonas, Bacteroides, Flavobacterium, Legionella, Serratia, and Stenotrophomonas has greatly increased interest in this class of enzymes (2). The fact that they hydrolyze almost all β-lactam antibiotics, including carbapenems, underlines their clinical relevance. In consequence, the potential spreading of these enzymes among pathogenic bacteria is a frightening possibility, which emphasizes the importance of understanding their properties. On the basis of the sequences, three subclasses of class B β-lactamases (B1 to B3) were identified, and a standard numbering scheme (BBL numbering) was proposed (13) by analogy to the ABL numbering scheme which has been widely used for class A β-lactamases. Due to the general low degree of identity between subclass sequences (<20%), classical alignment programs produce unreliable results. The proposed alignment (13) was facilitated by the availability of X-ray structures for B1 and B3 enzymes. Crystallographic structures have been described for several B1 enzymes: Bacillus cereus BcII (4, 11), Bacteroides fragilis CcrA (5, 8), Pseudomonas aeruginosa IMP-1 (7) and VIM-2 (unpublished data), and Chryseobacterium meningosepticum BlaB (14). Structural data are also available for two B3 enzymes: Stenotroptromonas maltophilia L1 (28) and Legionella gormanii FEZ-1 (15). Recently, we solved the first X-ray structure of a subclass B2 enzyme (CphA) produced by various species of Aeromonas (G. Garau, C. Bebrone, C. Anne, M. Galleni, J.-M. Frere, and O. Dideberg, unpublished data). Using all available three-dimensional structures, it is now possible to propose a bonafide structural alignment of the class B β-lactamases, and accordingly, to update the first proposed BBL scheme (Fig. ​(Fig.11). FIG. 1. Structural alignment of eight class B β-lactamases with known X-ray structures. The sequences are referred to by their familiar names. BCII, B. cereus 569H (16); IMP-1, P. aeruginosa 101/477 (17); CcrA, B. fragilis TAL3636 (25); VIM-2, P. aeruginosa ... For the three-dimensional structure comparison of the eight available structures, we used the program TOP (18) with the new option MAPS, allowing multiple alignments of protein structures. In addition, the program produces two ranking scores: the sequence identities of aligned residues and the structural diversity. The structural-diversity score was defined as RMS/(Nmatch/N0)3/2, where RMS is the root mean square deviation of the distances between matched Cα atoms, Nmatch is the number of matching residues, and (Nmatch/N0) is the matching fraction of two compared structures. N0 = (N1 + N2/2), where N1 and N2 are the numbers of amino acids in the two compared proteins. This score estimates the evolutionary distance between proteins. These two scores are shown in Table ​Table11 for all known X-ray structures. TABLE 1. Sequence identities of aligned residues and structure diversity among proteins Figure ​Figure11 displays the proposed alignment and numbering. Interestingly, the numbering of the important class B residues is conserved between old and new alignments. Improvements in the alignment concern mainly N and C termini and small shifts along the sequences. The main result of the new alignment is the identification of 14 sequence fragments of structurally conserved positions, which cover the entirety of all sequences (Fig. ​(Fig.1);1); they belong mainly to secondary-structure elements (α helices or β sheets). Notably, all Zn ligands are structurally aligned. The following comments can be made. (i) Only sequences of proteins of known structures are shown. (ii) For residues in lightface, the fact that they have the same number does not imply that they are structurally equivalent. (iii) For newly discovered enzymes, any insertion departing from the present numbering can be characterized by lowercase letters following the number of the last residue of the consensus sequence. Table ​Table22 shows the numbering of the putative zinc ligands. Not all proteins of known sequence are shown. Only enzymes with <50% sequence identity compared to the first reported sequence are included in the table. TABLE 2. Numbering of important class B residues In 1997, Neuwald et al. (23) detected a few proteins that have sequence similarities to (and may have given rise to) Zn-β-lactamases. They include enzymes with large variations in function (sulfatase; DNA cross-link repair enzyme) and which are encoded by yeast, plant, or bacterial open reading frames. Human glyoxalase II was also shown to belong to the superfamily. More recently, 17 groups with known functions were identified (9). In order to evaluate the structural diversity of the Zn-β-lactamase superfamily, human glyoxalase II (3) and rubredoxin oxygen-oxidoreductase from Desulfovibrio gigas (12) were also aligned using TOP, along with one member of each subclass. Table ​Table33 shows the sequence identities and structural diversity of the two proteins and BCII, CphA, and FEZ-1. As expected, low sequence identity corresponds to a high structural-diversity score. The structural-diversity scores for proteins belonging to a superfamily range from 1.4 to 2, in contrast to 3.5 to 4 for proteins with different folds (18). Interestingly and surprisingly, FEZ-1 is closer to glyoxalase II and rubredoxin oxygen-oxidoreductase than to BcII or CphA. TABLE 3. Sequence identities of aligned residues and structural diversity among proteins In the structural alignment, a large number of amino acid changes and insertions-deletions are observed. One hypothesis is that an ancient protein gave rise to the different subclasses of Zn-β-lactamases. A few candidates for the ancient protein are those related to essential biological functions within the cell, such as DNA or RNA processing or DNA repair (9). Nature used a limited number of scaffolds to generate a large variety of biological functions. Zn-β-lactamases are good examples of such a selection.

A catalytically silent FAAH-1 variant drives anandamide transport in neurons.

The endocannabinoid anandamide is removed from the synaptic space by a selective transport system, expressed in neurons and astrocytes, that remains molecularly uncharacterized. Here we describe a partly cytosolic variant of the intracellular anandamide-degrading enzyme fatty acid amide hydrolase-1 (FAAH-1), termed FAAH-like anandamide transporter (FLAT), that lacked amidase activity but bound anandamide with low micromolar affinity and facilitated its translocation into cells. Known anandamide transport inhibitors, such as AM404 and OMDM-1, blocked these effects. We also identified a competitive antagonist of the interaction of anandamide with FLAT, the phthalazine derivative ARN272, that prevented anandamide internalization in vitro, interrupted anandamide deactivation in vivo and exerted profound analgesic effects in rodent models of nociceptive and inflammatory pain, which were mediated by CB1 cannabinoid receptors. The results identify FLAT as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs.

Structural basis for the broad-spectrum inhibition of metallo-beta-lactamases by thiols

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.

Structure-Based Phylogeny of the Metallo-β-Lactamases

ABSTRACT The metallo-β-lactamases fall into two groups: Ambler class B subgroups B1 and B2 and Ambler class B subgroup B3. The two groups are so distantly related that there is no detectable sequence homology between members of the two different groups, but homology is clearly detectable at the protein structure level. The multiple structure alignment program MAPS has been used to align the structures of eight metallo-β-lactamases and five structurally homologous proteins from the metallo-β-lactamase superfamily, and that alignment has been used to construct a phylogenetic tree of the metallo-β-lactamases. The presence of genes from Eubacteria, Archaebacteria, and Eukaryota on that tree is consistent with a very ancient origin of the metallo-β-lactamase family.

Competitive inhibitors of the CphA metallo-beta-lactamase from Aeromonas hydrophila

ABSTRACT Various inhibitors of metallo-β-lactamases have been reported; however, none are effective for all subgroups. Those that have been found to inhibit the enzymes of subclass B2 (catalytically active with one zinc) either contain a thiol (and show less inhibition towards this subgroup than towards the dizinc members of B1 and B3) or are inactivators behaving as substrates for the dizinc family members. The present work reveals that certain pyridine carboxylates are competitive inhibitors of CphA, a subclass B2 enzyme. X-ray crystallographic analyses demonstrate that pyridine-2,4-dicarboxylic acid chelates the zinc ion in a bidentate manner within the active site. Salts of these compounds are already available and undergoing biomedical testing for various nonrelated purposes. Pyridine carboxylates appear to be useful templates for the development of more-complex, selective, nontoxic inhibitors of subclass B2 metallo-β-lactamases.

A Binding Site for Nonsteroidal Anti-inflammatory Drugs in Fatty Acid Amide Hydrolase.

In addition to inhibiting the cyclooxygenase (COX)-mediated biosynthesis of prostanoids, various widely used nonsteroidal anti-inflammatory drugs (NSAIDs) enhance endocannabinoid signaling by blocking the anandamide-degrading membrane enzyme fatty acid amide hydrolase (FAAH). The X-ray structure of FAAH in complex with the NSAID carprofen, along with site-directed mutagenesis, enzyme activity assays, and NMR analysis, has revealed the molecular details of this interaction, providing information that may guide the design of dual FAAH-COX inhibitors with superior analgesic efficacy.

β-Lactones Inhibit N-acylethanolamine Acid Amidase by S-Acylation of the Catalytic N-Terminal Cysteine

The cysteine amidase N-acylethanolamine acid amidase (NAAA) is a member of the N-terminal nucleophile class of enzymes and a potential target for anti-inflammatory drugs. We investigated the mechanism of inhibition of human NAAA by substituted β-lactones. We characterized pharmacologically a representative member of this class, ARN077, and showed, using high-resolution liquid chromatography-tandem mass spectrometry, that this compound forms a thioester bond with the N-terminal catalytic cysteine in human NAAA.

Structure of human N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: regulation of fatty acid ethanolamide biosynthesis by bile acids.

The fatty acid ethanolamides (FAEs) are lipid mediators present in all organisms and involved in highly conserved biological functions, such as innate immunity, energy balance, and stress control. They are produced from membrane N-acylphosphatidylethanolamines (NAPEs) and include agonists for G protein-coupled receptors (e.g., cannabinoid receptors) and nuclear receptors (e.g., PPAR-α). Here, we report the crystal structure of human NAPE-hydrolyzing phospholipase D (NAPE-PLD) at 2.65 Å resolution, a membrane enzyme that catalyzes FAE formation in mammals. NAPE-PLD forms homodimers partly separated by an internal ∼ 9-Å-wide channel and uniquely adapted to associate with phospholipids. A hydrophobic cavity provides an entryway for NAPE into the active site, where a binuclear Zn(2+) center orchestrates its hydrolysis. Bile acids bind with high affinity to selective pockets in this cavity, enhancing dimer assembly and enabling catalysis. These elements offer multiple targets for the design of small-molecule NAPE-PLD modulators with potential applications in inflammation and metabolic disorders.

Activity-Based Probe for N-Acylethanolamine Acid Amidase

N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α-amino-β-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo and to investigate the physiological and pathological roles of this enzyme.

Energy Landscapes Associated with Macromolecular Conformational Changes from Endpoint Structures

Conformational changes modulate macromolecular function by promoting the specific binding of ligands (such as in antigen recognition) or the stabilization of transition states in enzymatic reactions. However, quantitative characterization of the energetics underlying dynamic structural interconversions is still challenging and lacks a unified method. Here, we introduce a novel in silico approach based on the combined use of essential dynamics sampling and nonequilibrium free-energy calculations to obtain quantitative data on conformational energy landscapes. This technique allows the unbiased investigation of highly complex rearrangements, and does not require the crucial definition of user-defined collective variables. We show that free-energy values derived from profiles connecting the unliganded and ligand-bound X-ray structures of a bacterial nucleoside hydrolase match the experimental binding constant. This approach also provides first evidence for a rate-limiting character of the conformational transition in this enzyme, and an unexpected role of the protonation state of a single residue in regulating substrate binding and product release.