A zinc-binding motif conserved in glyoxalase II, β-lactamase and arylsulfatases

Melino, Sonia; Capo, Concetta; Dragani, Beatrice; Aceto, Antonio; Petruzzelli, Raffaele

doi:10.1016/s0968-0004(98)01264-x

Cited by 63 publications

(64 citation statements)

References 11 publications

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“…Nitrocefin (26) has been extensively used in mechanistic investigations of metallo-␤-lactamase action (11,12,27), as both the loss of substrate and appearance of product are accompanied by substantial changes in absorbance ( max ϳ390 and 482 nm, respectively). It has further been shown that nitrocefin hydrolysis by L1 proceeds through a transiently populated intermediate with a strong absorbance at 665 nm, the breakdown of which coincides with appearance of product (12).…”

Section: Resultsmentioning

confidence: 99%

“…We have previously proposed (15) that L1 hydrolyzes substrate through a tetrahedral oxyanion intermediate; however, pre-steady-state kinetic experiments using nitrocefin as substrate (12) show that the major intermediate has the same characteristics as that observed for the CcrA enzyme (11). Nitrocefin ( Fig.…”

mentioning

confidence: 84%

“…Several models of such a reaction scheme have been postulated (7,10,14,15); these vary primarily in the source of the proton required for product formation. Experimental evidence points to different conclusions for different enzymes: in the BCII enzyme from Bacillus cereus, a conserved aspartate in the active site appears to shuttle a proton from the oxyanion to the amide nitrogen, and the major intermediate is a dianionic species (10), whereas in CcrA from Bacteriodes fragilis, the major intermediate in hydrolysis of the colorimetric ␤-lactam nitrocefin is the deprotonated product, and the rate-determining step is donation of a proton by a second zinc-bound water (11). This apparent mechanistic heterogeneity between different metallo-␤-lactamases is also manifest in the results of attempts at inhibitor design (16 -19), in which many compounds show a wide variation in potency against the different enzymes, highlighting the existence of subtle variations in active site structure within the family and thus need for detailed study of the individual proteins.…”

mentioning

confidence: 99%

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Novel Mechanism of Hydrolysis of Therapeutic β-Lactams byStenotrophomonas maltophilia L1 Metallo-β-lactamase

Spencer¹,

Clarke²,

Walsh³

2001

Journal of Biological Chemistry

100

152

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Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of ␤-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-␤-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark intermediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the ␤-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the ␤-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-␤-lactamases may be atypical and that cleavage of the ␤-lactam amide bond is the rate-determining step for breakdown of the majority of ␤-lactam substrates by the L1 enzyme.Zinc-dependent or metallo-␤-lactamases (1) are bacterial enzymes of considerable clinical and mechanistic interest. In the clinical context, their ability to hydrolyze the newer generation of carbapenem antibiotics (2), together with the potential for interspecies transfer arising from their presence on mobile genetic elements (3, 4), is a cause for increasing concern as carbapenem use becomes more widespread. The group includes both nonspecific enzymes (able to efficiently hydrolyze penicillin and cephalosporin antibiotics in addition to carbapenems) and specific enzymes (5) and may utilize one or two zinc ions for maximal activity (6 -8). Although mechanistic analogies with zinc peptidases have been attempted (7), it is now clear that metallo-␤-lactamases are members of a distinct superfamily of hydrolases that includes bacterial arylsulfatases and type II glyoxalases (8, 9). Despite increasing attention in recent years (10 -12), understanding of metallo-␤-lactamase mechanism remains incomplete, and there are to date no clinically effective inhibitors.There is general agreement that metallo-␤-lactamases hydrolyze substrate by a nucleophilic attack of zinc-bound water upon the carbonyl carbon of the scissile bond (Ref. 13 and references therein). This would be expected to lead to formation of a tetrahedral oxyanion intermediate that would decay to product via cleavage of the ␤-lactam amide bond and protonation of the amide nitrogen. Several models of such a reaction scheme have been postulated (7,10,14,15); these vary primarily in the source of the proton required for product formation. Experimental evidence points to different conclusions for different enzymes: in the BCII enzyme from Bacillus cereus, a conserved aspartate in the active site appears to shuttle a proton from the oxyanion to the amide nitrogen, and the major intermedia...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 84%

mentioning

confidence: 99%

See 1 more Smart Citation

Novel Mechanism of Hydrolysis of Therapeutic β-Lactams byStenotrophomonas maltophilia L1 Metallo-β-lactamase

Spencer¹,

Clarke²,

Walsh³

2001

Journal of Biological Chemistry

100

152

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“…The samples were assayed for AI activity as described (3). and arylsulfatase (30). Sequence alignment of the second region locates one invariable histidine (H169) among AiiA and the three metalloenzymes (Fig.…”

Section: Methodsmentioning

confidence: 99%

AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora

Dong

Xu²,

Li³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

745

322

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N -acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum , several Erwinia species, and the human pathogen Pseudomonas aeruginosa . A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene ( aiiA ) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a “HXHXDH” zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.

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“…Based on its similarity to the metallo-␤-lactamases, we predicted that glyoxalase II binds two Zn(II) ions, utilizing five histidines, two aspartic acids, and a bridging water molecule (18). These predictions have been restated by Melino et al (22) and supported through the recent determination of the human glyoxalase II crystal structure (23). The crystallographic data indicates that the metal binding and active sites of glyoxalase II resemble those in the metallo-␤-lactamase L1 from Stenotrophomonas maltophilia (24).…”

mentioning

confidence: 86%

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

Zang

Hollman

Crawford

et al. 2001

Journal of Biological Chemistry

127

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Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or antiprotozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.The glyoxalase system consists of two enzymes that convert cytotoxic 2-oxoaldehydes into hydroxy acids utilizing the cofactor glutathione. Under physiological conditions, glyoxalase I (lactoylglutathione lyase) catalyzes the formation of S-D-lactoylglutathione (SLG) 1 in the presence of methylglyoxal and glutathione (1). Methylglyoxal, a cytotoxic compound that can inactivate proteins, modify guanylate residues in DNA, and create interstrand cross-links (2-4), is formed as a by-product of several biochemical reactions including that of triose-phosphate isomerase (2). Glyoxalase II (hydroxyacylglutathione hydrolase) hydrolyzes SLG to form D-lactic acid and regenerate glutathione (1). SLG is also known to exhibit cytotoxic effects through the inhibition of DNA synthesis (5). Therefore, the glyoxalase system is thought to play a major role in chemical detoxification (5, 6).The glyoxalase system has been the subject of intense study because of its potential implications in anti-protozoal and antitumor drug design (6 -8). Increased cellular levels of methylglyoxal and glyoxalase activity are associated with tumor cells, the malaria parasite, and several complications associated with diabetes (6). Inhibitors of glyoxalase I and glyoxalase II have been designed as potential anti-tumor agents; however, all inhibitors tested to date exhibited problems that limited their therapeutic usefulness (6 -9). It should be noted that these inhibition studies were all conducted with little or no information on the active site structure or the kinetic mechanism of the enzyme. Therefore, it is clear that a more detailed understanding of the active site of glyoxalase II and its reaction mechanism is essential to the rational design an...

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A zinc-binding motif conserved in glyoxalase II, β-lactamase and arylsulfatases

Cited by 63 publications

References 11 publications

Novel Mechanism of Hydrolysis of Therapeutic β-Lactams byStenotrophomonas maltophilia L1 Metallo-β-lactamase

Novel Mechanism of Hydrolysis of Therapeutic β-Lactams byStenotrophomonas maltophilia L1 Metallo-β-lactamase

AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

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