Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and ␤-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of ␤-lactamases and DD-peptidases. The chemical reactivities of esters and ␤-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying ␤-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C ␤-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "␤-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to ␤-lactamases, EstB shows no ␤-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous ␤-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus ␤-lactam cleavage.

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.Keywords: tryptophan fluorescence lifetime; fluorescence quenching; disulphide (disulfide) bridges; photochemical reaction; protein structure damaged by UV light; SS bond disruption; indole; theoretical model Two divergent theories of the mechanisms involved in ultraviolet (UV) inactivation of enzymes have been developed over a period of years. One theory proposes that the random destruction of any amino acid residue causes inactivation (Augenstein and Riley 1964). The second emphasizes the importance of the disruption of a cluster of specific cystine residues (cysteines involved in disulphide bridges; Augenstein and Riley 1964). It was also found that the effective destruction of cystine, tryptophan, tyrosine, and phenylalanine occurs on UV irradiation of proteins (Kazutomo

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The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (K a % 10 5 M 21 ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. TheAbbreviations: AA, all-atom; AoC, Aspergillus oryzae cutinase; AOT, sodium bis(2-ethylhexyl) sulfosuccinate; cmc, critical micelle concentration; DEP, diethylphosphate; DNPP, diethyl p-nitrophenyl phosphate; EDTA, ethylene diamine tetraacetate; FsC, Fusarium solani cutinase; GcC, Glomerella cingulata cutinase; HiC, Humicola insolens cutinase; HSQC, heteronuclear singlequantum coherence; IPTG, isopropyl b-D-1-thiogalactopyranoside; ITC, isothermal titration calorimetry; MD, molecular dynamics; MEP, monoethylphosphate; MWCO, molecular weight cut-off; PAGE, polyacrylamide gel electrophoresis; RMSD, root mean square deviation; RMSF, root mean square fluctuation; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; SANS, small-angle neutron scattering; SDS, sodium dodecyl sulfate.Additional Supporting Information may be found in the online version of this article.An interactive view is available in the electronic version of the article. enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.