Natural product-guided discovery of a fungal chitinase inhibitor

Natural products are often large, synthetically intractable molecules, yet frequently offer surprising inroads into previously unexplored chemical space for enzyme inhibitors. Argifin is a cyclic pentapeptide that was originally isolated as a fungal natural product. It competitively inhibits family 18 chitinases by mimicking the chitooligosaccharide substrate of these enzymes. Interestingly, argifin is a nanomolar inhibitor of the bacterial-type subfamily of fungal chitinases that possess an extensive chitin-binding groove, but does not inhibit the much smaller, plant-type enzymes from the same family that are involved in fungal cell division and are thought to be potential drug targets. Here we show that a small, highly efficient, argifin-derived, nine-atom fragment is a micromolar inhibitor of the plant-type chitinase ChiA1 from the opportunistic pathogen Aspergillus fumigatus. Evaluation of the binding mode with the first crystal structure of an A. fumigatus plant-type chitinase reveals that the compound binds the catalytic machinery in the same manner as observed for argifin with the bacterial-type chitinases. The structure of the complex was used to guide synthesis of derivatives to explore a pocket near the catalytic machinery. This work provides synthetically tractable plant-type family 18 chitinase inhibitors from the repurposing of a natural product.     

Atmospheric chemistry of n-C(x)F(2)(x)(+1)CHO (x = 1, 2, 3, 4): fate of n-C(x)F(2)(x)(+1)C(O) radicals

Smog chamber/FTIR techniques were used to study the atmospheric fate of n-C(x)F(2)(x)(+1)C(O) (x = 1, 2, 3, 4) radicals in 700 Torr O(2)/N(2) diluent at 298 +/- 3 K. A competition is observed between reaction with O(2) to form n-C(x)()F(2)(x)()(+1)C(O)O(2) radicals and decomposition to form n-C(x)F(2)(x)(+1) radicals and CO. In 700 Torr O(2)/N(2) diluent at 298 +/- 3 K, the rate constant ratio, k(n-C(x)F(2)(x)(+1)C(O) + O(2) --> n-C(x)F(2)(x)(+1)C(O)O(2))/k(n-C(x)F(2)(x)(+1)C(O) --> n-C(x)F(2)(x)(+1) + CO) = (1.30 +/- 0.05) x 10(-17), (1.90 +/- 0.17) x 10(-19), (5.04 +/- 0.40) x 10(-20), and (2.67 +/- 0.42) x 10(-20) cm(3) molecule(-1) for x = 1, 2, 3, 4, respectively. In one atmosphere of air at 298 K, reaction with O(2) accounts for 99%, 50%, 21%, and 12% of the loss of n-C(x)F(2)(x)(+1)C(O) radicals for x = 1, 2, 3, 4, respectively. Results are discussed with respect to the atmospheric chemistry of n-C(x)F(2)(x)(+1)C(O) radicals and their possible role in contributing to the formation of perfluorocarboxylic acids in the environment.     

Oxo transfer reactions mediated by bis(dithiolene)tungsten analogues of the active sites of molybdoenzymes in the DMSO reductase family: comparative reactivity of tungsten and molybdenum.

The discovery of tungsten enzymes and molybdenum/tungsten isoenzymes, in which the mononuclear catalytic sites contain a metal chelated by one or two pterin-dithiolene cofactor ligands, has lent new significance to tungsten-dithiolene chemistry. Reaction of [W(CO)(2)(S(2)C(2)Me(2))(2)] with RO(-) affords a series of square pyramidal desoxo complexes [W(IV)(OR')(S(2)C(2)Me(2))(2)](1)(-), including R' = Ph (1) and Pr(i)() (3). Reaction of 1 and 3 with Me(3)NO gives the cis-octahedral complexes [W(VI)O(OR')(S(2)C(2)Me(2))(2)](1)(-), including R' = Ph (6) and Pr(i)() (8). These W(IV,VI) complexes are considered unconstrained versions of protein-bound sites of DMSOR and TMAOR (DMSOR = dimethylsulfoxide reductase, TMAOR = trimethylamine N-oxide reductase) members of the title enzyme family. The structure of 6 and the catalytic center of one DMSO reductase isoenzyme have similar overall stereochemistry and comparable bond lengths. The minimal oxo transfer reaction paradigm thought to apply to enzymes, W(IV) + XO --> W(VI)O + X, has been investigated. Direct oxo transfer was demonstrated by isotope transfer from Ph(2)Se(18)O. Complex 1 reacts cleanly and completely with various substrates XO to afford 6 and product X in second-order reactions with associative transition states. The substrate reactivity order with 1 is Me(3)NO > Ph(3)AsO > pyO (pyridine N-oxide) > R(2)SO > Ph(3)PO. For reaction of 3 with Me(3)NO, k(2) = 0.93 M(-)(1) s(-)(1), and for 1 with Me(2)SO, k(2) = 3.9 x 10(-)(5) M(-)(1) s(-)(1); other rate constants and activation parameters are reported. These results demonstrate that bis(dithiolene)W(IV) complexes are competent to reduce both N-oxides and S-oxides; DMSORs reduce both substrate types, but TMAORs are reported to reduce only N-oxides. Comparison of k(cat)/K(M) data for isoenzymes and k(2) values for isostructural analogue complexes reveals that catalytic and stoichiometric oxo transfer, respectively, from substrate to metal is faster with tungsten and from metal to substrate is faster with molybdenum. These results constitute a kinetic metal effect in direct oxo transfer reactions for analogue complexes and for isoenzymes provided the catalytic sites are isostructural. The nature of the transition state in oxo transfer reactions of analogues is tentatively considered. This research presents the first kinetics study of substrate reduction via oxo transfer mediated by bis(dithiolene)tungsten complexes.     

Studies of iron(II) and iron(III) complexes with fac-N2O, cis-N2O2 and N2O3 donor ligands: models for the 2-His 1-carboxylate motif of non-heme iron monooxygenases

Enzymes in the oxygen-activating class of mononuclear non-heme iron oxygenases (MNOs) contain a highly conserved iron center facially ligated by two histidine nitrogen atoms and one carboxylate oxygen atom that leave one face of the metal center (three binding sites) open for coordination to cofactor, substrate, and/or dioxygen. A comparative family of [Fe(II/III)(N(2)O(n))(L)(4-n))](±x), n = 1-3, L = solvent or Cl(-), model complexes, based on a ligand series that supports a facially ligated N,N,O core that is then modified to contain either one or two additional carboxylate chelate arms, has been structurally and spectroscopically characterized. EPR studies demonstrate that the high-spin d(5) Fe(III)g = 4.3 signal becomes more symmetrical as the number of carboxylate ligands decreases across the series Fe(N(2)O(3)), Fe(N(2)O(2)), and Fe(N(2)O(1)), reflecting an increase in the E/D strain of these complexes as the number of exchangeable/solvent coordination sites increases, paralleling the enhanced distribution of electronic structures that contribute to the spectral line shape. The observed systematic variations in the Fe(II)-Fe(III) oxidation-reduction potentials illustrate the fundamental influence of differential carboxylate ligation. The trend towards lower reduction potential for the iron center across the [Fe(III)(N(2)O(1))Cl(3)](-), [Fe(III)(N(2)O(2))Cl(2)](-) and [Fe(III)(N(2)O(3))Cl](-) series is consistent with replacement of the chloride anions with the more strongly donating anionic O-donor carboxylate ligands that are expected to stabilize the oxidized ferric state. This electrochemical trend parallels the observed dioxygen sensitivity of the three ferrous complexes (Fe(II)(N(2)O(1)) < Fe(II)(N(2)O(2)) < Fe(II)(N(2)O(3))), which form μ-oxo bridged ferric species upon exposure to air or oxygen atom donor (OAD) molecules. The observed oxygen sensitivity is particularly interesting and discussed in the context of α-ketoglutarate-dependent MNO enzyme mechanisms.     

Lysozymes: a chapter of molecular biology

Hen egg-white lysozyme was the first enzyme whose tertiary structure could be elucidated. The peptide chain of this enzyme is arranged in two sections, of approximately equal size, that are separated by a deep cleft. Substrates (and inhibitors) are bound in this cleft via hydrogen bonds and are hydrolyzed under the action of Glu 35 and Asp 52, which form the active site of the enzyme. Although the lysozymes, which occur in many species of animals and plants, exhibit differences in their chemical behavior, they have the same qualitative biological activity; quantitatively important differences have been noted which also concern the specificity. Infection of E. coli with bacteriophages gives rise to a lysozyme whose formation is controlled by the phage DNA. The fact that mutated lysozymes are produced when the phages are treated with mutagens opens new fields of research in molecular biology.     

Oxygen activation by nonheme iron(II) complexes: alpha-keto carboxylate versus carboxylate

Mononuclear iron(II) alpha-keto carboxylate and carboxylate compounds of the sterically hindered tridentate face-capping ligand Tp(Ph2) (Tp(Ph2) = hydrotris(3,5-diphenylpyrazol-1-yl)borate) were prepared as models for the active sites of nonheme iron oxygenases. The structures of an aliphatic alpha-keto carboxylate complex, [Fe(II)(Tp(Ph2))(O(2)CC(O)CH(3))], and the carboxylate complexes [Fe(II)(Tp(Ph2))(OBz)] and [Fe(II)(Tp(Ph2))(OAc)(3,5-Ph(2)pzH)] were determined by single-crystal X-ray diffraction, all of which have five-coordinate iron centers. Both the alpha-keto carboxylate and the carboxylate compounds react with dioxygen resulting in the hydroxylation of a single ortho phenyl position of the Tp(Ph2) ligand. The oxygenation products were characterized spectroscopically, and the structure of the octahedral iron(III) phenolate product [Fe(III)(Tp(Ph2))(OAc)(3,5-Ph(2)pzH)] was established by X-ray diffraction. The reaction of the alpha-keto carboxylate model compounds with oxygen to produce the phenolate product occurs with concomitant oxidative decarboxylation of the alpha-keto acid. Isotope labeling studies show that (18)O(2) ends up in the Tp(Ph2) phenolate oxygen and the carboxylate derived from the alpha-keto acid. The isotope incorporation mirrors the dioxygenase nature of the enzymatic systems. Parallel studies on the carboxylate complexes demonstrate that the oxygen in the hydroxylated ligand is also derived from molecular oxygen. The oxygenation of the benzoylformate complex is demonstrated to be first order in metal complex and dioxygen, with activation parameters DeltaH++ = 25 +/- 2 kJ mol(-1) and DeltaS++ = -179 +/- 6 J mol(-1) K(-1). The rate of appearance of the iron(III) phenolate product is sensitive to the nature of the substituent on the benzoylformate ligand, exhibiting a Hammett rho value of +1.3 indicative of a nucleophilic mechanism. The proposed reaction mechanism involves dioxygen binding to produce an iron(III) superoxide species, nucleophilic attack of the superoxide at the alpha-keto functionality, and oxidative decarboxylation of the adduct to afford the oxidizing species that attacks the Tp(Ph2) phenyl ring. Interestingly, the alpha-keto carboxylate complexes react 2 orders of magnitude faster than the carboxylate complexes, thus emphasizing the key role that the alpha-keto functionality plays in oxygen activation by alpha-keto acid-dependent iron enzymes.     

HYDROPEROXYNAPHTHALIMIDE DERIVATIVE-MEDIATED OXIDATION OF LYSOZYME

Abstract— Lysozyme was photoirradiated in the presence of photo-Fenton reagents (hydroperoxynaph-thalimide derivatives) at 366 nm. Enzymatic activities of photoirradiated lysozymes were lower than that of native lysozyme. Taking account of the results of amino acid analysis and of fluorescence spectra, it was probably that Trp residues in the photoirradiated lysozyme were oxidized with hydroxyl radicals. The reagents formed complexes with lysozyme as proved by the inhibitory effects of the reagents on the enzymatic activities ( K ₁= 4.7 ± 1.2 × 10⁴ M for HPO II, a hydroperoxide derivative of naphthalimide), which suggested that these reagents were bound to the active site cleft of lysozyme, and the Trp residues located in or near the active site cleft were photooxidized. Fluorescence-difference spectra of photoirradiated lysozymes showed that Trp 62 was preferentially photooxidized. Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and circular dichroism spectra showed that the photooxidation examined here induced no significant change in the molecular size but a slight change in the conformation of lysozyme, which suggests the usefulness of the reagents in the site-selective oxidation of biopolymers.     

Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

Background  

β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system.     

Palladium catalyzed carboxylation of allylstannanes and boranes using CO2

A family of well-defined (η(3)-allyl)Pd(L)(carboxylate) (L = PR(3) or NHC) complexes are by far the most efficient catalysts reported to date for the catalytic carboxylation of allylstannanes into allylcarboxylates using CO(2). The substrate scope of this reaction is extended to both substituted allylstannanes and allylboranes.     

Atmospheric chemistry of perfluoroaldehydes (CxF2x+1CHO) and fluorotelomer aldehydes (CxF2x+1CH2CHO): quantification of the important role of photolysis

The UV absorption spectra of CF(3)CHO, C(2)F(5)CHO, C(3)F(7)CHO, C(4)F(9)CHO, CF(3)CH(2)CHO, and C(6)F(13)CH(2)CHO were recorded over the range 225-400 nm at 249-297 K. C(x)F(2)(x)(+1)CHO and C(x)F(2)(x)(+1)CH(2)CHO have broad absorption features centered at 300-310 and 290-300 nm, respectively. The strength of the absorption increases with the size of the C(x)F(2)(x)(+1) group. There was no discernible (<5%) effect of temperature on the UV spectra. Quantum yields for photolysis at 254 and 308 nm were measured. Quantum yields at 254 nm were 0.79 +/- 0.09 (CF(3)CHO), 0.81 +/- 0.09 (C(2)F(5)CHO), 0.63 +/- 0.09 (C(3)F(7)CHO), 0.60 +/- 0.09 (C(4)F(9)CHO), 0.74 +/- 0.08 (CF(3)CH(2)CHO), and 0.55 +/- 0.09 (C(6)F(13)CH(2)CHO). Quantum yields at 308 nm were 0.17 +/- 0.03 (CF(3)CHO), 0.08 +/- 0.02 (C(4)F(9)CHO), and 0.04 +/- 0.01 (CF(3)CH(2)CHO). The quantum yields decrease with increasing size of the C(x)F(2)(x)(+1) group and with increasing wavelength of the photolysis light. The photolysis quantum yield at 308 nm for CF(3)CHO measured here is a factor of at least 8 greater than that reported previously. Photolysis is probably the dominant atmospheric fate of C(x)F(2)(x)(+1)CHO (x = 1-4) and is an important fate of C(x)F(2)(x)(+1)CH(2)CHO (x = 1 and 6). These results have important ramifications concerning the yield of perfluorocarboxylic acids in the atmospheric oxidation of fluorotelomer alcohols.     

Spectroscopic and computational studies of α-keto acid binding to Dke1: understanding the role of the facial triad and the reactivity of β-diketones

The O(2) activating mononuclear nonheme iron enzymes generally have a common facial triad (two histidine and one carboxylate (Asp or Glu) residue) ligating Fe(II) at the active site. Exceptions to this motif have recently been identified in nonheme enzymes, including a 3His triad in the diketone cleaving dioxygenase Dke1. This enzyme is used to explore the role of the facial triad in directing reactivity. A combination of spectroscopic studies (UV-vis absorption, MCD, and resonance Raman) and DFT calculations is used to define the nature of the binding of the α-keto acid, 4-hydroxyphenlpyruvate (HPP), to the active site in Dke1 and the origin of the atypical cleavage (C2-C3 instead of C1-C2) pattern exhibited by this enzyme in the reaction of α-keto acids with dioxygen. The reduced charge of the 3His triad induces α-keto acid binding as the enolate dianion, rather than the keto monoanion, found for α-keto acid binding to the 2His/1 carboxylate facial triad enzymes. The mechanistic insight from the reactivity of Dke1 with the α-keto acid substrate is then extended to understand the reaction mechanism of this enzyme with its native substrate, acac. This study defines a key role for the 2His/1 carboxylate facial triad in α-keto acid-dependent mononuclear nonheme iron enzymes in stabilizing the bound α-keto acid as a monoanion for its decarboxylation to provide the two additional electrons required for O(2) activation.     

Syntheses of 1-alkyl-1,2,4-triazoles and the formation of quaternary 1-alkyl-4-polyfluoroalkyl-1,2,4-triazolium salts leading to ionic liquids

1,2,4-triazole was alkylated (alkyl = methyl, butyl, heptyl, decyl) at N-1 in >90% isolated yields. The resulting 1-alkyl triazoles were quaternized at N-4 in >98% isolated yields using fluorinated alkyl halides with >98% isolated yields, under neat reaction conditions at 100-120 degrees C to form N1-CH(3)-N4-(CH(2))(2)C(m)F(2)(m)(+ 1)-triazolium (Taz) iodide (m = 1, 6), N1-C(4)H(9)-N4-(CH(2))(2)C(m)F(2)(m)(+ 1)-Taz iodide (m = 1, 4, 6), N1-C(7)H(15)-N4-(CH(2))(2)C(m)F(2)(m)(+ 1)-Taz iodide (m = 1, 4, 6), N1-C(10)H(21)-N4-(CH(2))(2)C(m)F(2)(m)(+1)-Taz iodide (m = 1, 4), and N1-C(n)H(2)(n )(+ 1)-N4-(CH(2))(2)F-Taz bromide (n = 4, 7, 10). Single-crystal X-ray analyses confirmed the structure of [1-CH(3)-4-CH(2)CH(2)CF(3)-Taz](+)I(-). It crystallized in the orthorhombic space group Pccn, and the unit cell dimensions were a = 13.8289(9) A, b = 17.3603(11) A, c = 9.0587(6) A (alpha = beta = gamma = 90 degrees ). Metathesis of these polyfluoroalkyl-substituted triazolium halides with other salts led to the formation of quaternary compounds, some of which comprise ionic liquids, namely, [R(R(f))-Taz](+)Y(-) (Y = NTf(2), BF(4), PF(6), and OTf), in good isolated yields without the need for further purification: N1-CH(3)-N4-(CH(2))(2)C(m)F(2)(m)( +) (1)-Taz Y (m = 1, 6; Y = NTf(2)), N1-C(4)H(9)-N4-(CH(2))(2)C(m)F(2)(m)(+ 1)-Taz Y (m = 1, 4, 6; Y = NTf(2)), N1- C(7)H(15)-N4-(CH(2))(2)C(m)F(2)(m)(+ 1)-Taz Y (m = 1, 4, 6; Y = NTf(2)), N1-C(10)H(21)-N4-(CH(2))(2)C(m)F(2)(m)(+1)-Taz Y (n = 1, 4; Y = NTf(2)), N1-C(n)H(2)(n )(+ 1)-N4-(CH(2))(2)F-Taz Y (n = 7, 10; Y = NTf(2)), N1-C(10)H(21)-N4-(CH(2))(2)F-TazY (Y = OTf), N1-C(7)H(15)-N4-(CH(2))(2)F-TazY (Y = BF(4)), N1-C(4)H(9)-N4-(CH(2))(2)C(m)F(2)(m) (+ 1)-Taz Y (m = 4, 6; Y = PF(6)), N1-C(7)H(15)-N4-(CH(2))(2)C(4)F(9)-Taz Y (Y = PF(6)), N1-C(4)H(9)-N4-(CH(2))(2)C(m)F(2)(m)(+ 1)-Taz Y (m = 4, 6; Y = OTf). All new compounds were characterized by (1)H, (19)F, and (13)C NMR and MS spectra and elemental analyses. T(g)s and T(m)s of ionic liquids were determined by DSC.     

Water-soluble substrates of the peptidoglycan-modifying enzyme O-acetylpeptidoglycan esterase (Ape1) from Neisseria gonorrheae

Peptidoglycan is the component of the bacterial cell wall that is essential for maintaining the shape and rigidity of the cell. As such, its polymeric structure, consisting of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), is also a target for the action of host defense enzymes, such as lysozymes. Many bacteria have developed methods of masking their cell wall from these environmental dangers through the addition of aglycon moieties that prevent recognition or sterically hinder the degradative action of exogenous enzymes that would otherwise prove detrimental to the cell. Peptidoglycan acetyl-transferases (Pat's) and O-acetylpeptidoglycan esterases (Ape's) are the enzymes responsible for the controlled addition and removal of acetate onto the C-6 hydroxyl group of MurNAc residues in peptidoglycan. Studies on Ape1, an O-acetylpeptidoglycan esterase found in Neisseria gonorrheae, have suggested that this enzyme is essential for bacterial viability and thus presents an attractive target for antibacterial design. Previous studies on Ape1 have been hindered by the fact that Ape1's natural substrate is an insoluble polymer. In this paper we outline the design, synthesis, and testing of the water-soluble di- and monosaccharide substrate analogues 1 and 2. Both 1 and 2 serve as substrates of Ape1 with k(cat)/K(M) values of (5.1 ± 1.7) × 10(3) M(-1) s(-1) and (3.1 ± 0.8) × 10(3) M(-1) s(-1), respectively. It was determined that the substitution of the GlcNAc residue in compound 1 with an O-benzyl group in compound 2 did not significantly decrease the enzyme's affinity for the monosaccharide. These findings are important as they demonstrate that the catalytic prowess of Ape1 is not dependent on its binding to a polymeric substrate. This ensures that small molecule transition state/intermediate analogues can also capture the transition state binding energy of Ape1 and potentially serve as potent inhibitors. The synthetic route to compounds 1 and 2 could readily be modified to allow for the installation of a wide variety of functional groups at the MurNAc C-6 position in both the mono- and disaccharide scaffolds. This will serve as a general method for the construction of Ape1 substrates and inhibitors.     

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity.

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.     

Modification of Cyclodextrins for Use as Artificial Enzymes

The magical powers of enzymes have been attributed to their ability to bind specific substrates and catalyze reactions of the bound substrate. Artificial enzymes synthetically mimic the binding and the catalytic site to produce molecules that are not only smaller in size but also potentially have similar activity to the real enzymes. The main objective of our research is to create artificial redox enzymes by using cyclodextrins as binding sites and attaching flavin derivatives as the catalytic site. We have developed a strategy to attach a catalytic site to cyclodextrin exclusively at the 2-, 3- or the 6-position. The evaluation of the artificial enzyme in which flavin is attached to the 2-position gives a 647-fold acceleration factor. Although this is modest compared to those of real enzymes (which can have acceleration factors of a trillion), the artificial enzymes allow us to understand the elements that contribute to the incredible catalytic power of enzymes.     

Inhibition of Human Immunodeficiency Type 1 Virus (HIV-1) Life Cycle by Different Egg White Lysozymes

Lysozyme is a relatively small enzyme with different biological activities, which is found in tears, saliva, egg white, and human milk. In the study, the anti-HIV-1 activity of lysozymes purified from quail, Meleagris, and hen egg white has been determined. For this end, a time-of-drug-addition assay was performed to identify the target of anti-HIV-1 agents and for determination of probable anti HIV-1 mechanism of the studied lysozyme, the binding affinity of the lysozymes to the human CD4 receptor was studied by molecular docking method. To define structural differences between studied lysozymes, structural motifs of them were predicted by MEME tool. Quail, hen, and Meleagris lysozymes showed potent anti-HIV-1 activity with EC₅₀ of 7.5, 10, and 55 nM, respectively. The time-of-drug-addition study demonstrated that the inhibitory effect of all purified lysozymes is before HIV-1 infection. The frequency and intensity of CD4 expression in PBMCs decreased in the presence of all mentioned lysozymes. Also, the expression level of C-C chemokine receptor type 5 (CCR5) and chemokine receptor type 4 (CXCR4) on CD4+ T cells was not changed in cells treated with these lysozymes. The results of in silico study confirmed that the binding energy of quail lysozyme with CD4 was more than that of other studied lysozymes. The results revealed that these lysozymes restrict HIV-1 attachment to host cell CD4.     

Alpha/Beta-hydrolase fold enzymes: structures,functions and mechanisms

The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate-assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined. Relationships between active site anatomy, topology and conformational rearrangements in the protein molecule is discussed in the context of enzyme mechanism of action.     

Structure of Saccharomyces cerevisiae chitinase 1 and screening-based discovery of potent inhibitors

Chitinases hydrolyse the beta(1,4)-glycosidic bonds of chitin, an essential fungal cell wall component. Genetic data on a subclass of fungal family 18 chitinases have suggested a role in cell wall morphology. Specific inhibitors of these enzymes would be useful as tools to study their role in cell wall morphogenesis and could possess antifungal properties. Here, we describe the crystallographic structure of a fungal "plant-type" family 18 chitinase, that of Saccharomyces cerevisiae CTS1. The enzyme is active against 4-methylumbelliferyl chitooligosaccharides and displays an unusually low pH optimum for activity. A library screen against ScCTS1 yielded hits with Ki 's as low as 3.2 microM. Crystal structures of ScCTS1 in complex with inhibitors from three series reveal striking mimicry of carbohydrate substrate by small aromatic moieties and a pocket that could be further exploited in optimization of these inhibitors.     

Polymer-anchored peroxo compounds of vanadium(V) and molybdenum(VI): synthesis, stability, and their activities with alkaline phosphatase and catalase

We generated a series of new polymer-bound peroxo complexes of vanadium(V) and molybdenum(VI) of the type [VO(O(2))(2)(sulfonate)]-PSS [PSS = poly(sodium 4-styrene sulfonate)] (PV(3)), [V(2)O(2)(O(2))(4)(carboxylate)VO(O(2))(2)(sulfonate)]-PSSM [PSSM = poly(sodium styrene sulfonate-co-maleate)] (PV(4)), [Mo(2)O(2)(O(2))(4)(carboxylate)]-PA [PA = poly(sodium acrylate)] (PMo(1)), [MoO(O(2))(2)(carboxylate)]-PMA [PMA = poly(sodium methacrylate)] (PMo(2)), and [MoO(O(2))(2)(amide)]-PAm [PAm = poly(acrylamide)] (PMo(3)) by reacting V(2)O(5) (for PV(3) and PV(4)) or H(2)MoO(4) (for PMo(1), PMo(2), and PMo(3)) with H(2)O(2) and the respective water-soluble macromolecular ligand at pH 5-6. The compounds were characterized by elemental analysis (CHN and energy-dispersive X-ray spectroscopy), spectral studies (UV-vis, IR, (13)C NMR, (51)V NMR, and (95) Mo NMR), thermal (TGA) as well as scanning electron micrographs (SEM), and EDX analysis. It has been demonstrated that compounds retain their structural integrity in solutions of a wide range of pH values and are approximately 100 times weaker as substrate to the enzyme catalase relative to H(2)O(2), its natural substrate. The effect of the title compounds, along with previously reported compounds [V(2)O(2)(O(2))(4)(carboxylate)]-PA (PV(1)) and [VO(O(2))(2)(carboxylate)]-PMA (PV(2)) on rabbit intestine alkaline phosphatase (ALP) has been investigated and compared with the effect induced by the free diperoxometallates viz. Na[VO(O(2))(2)(H(2)O)] (DPV), [MoO(O(2))(2)(glycine)(H(2)O)] (DMo(1)), and [MoO(O(2))(2)(asparagine)(H(2)O)] (DMo(2)). It has been observed that although all the compounds tested are potent inhibitors of the enzyme, the polymer-bound and neat complexes act via distinct mechanisms. Each of the macromolecular compounds is a classical noncompetitive inhibitor of ALP. In contrast, the action of neat pV and heteroligand pMo compounds on the enzyme function is consistent with a mixed type of inhibition.     

Covalent attachment of lysozyme to cotton/cellulose materials: protein verses solid support activation

Covalent attachment of enzymes to cellulosic materials like cotton is of interest where either release or loss of enzyme activity over time needs to be avoided. The covalent attachment of an enzyme to a cellulosic substrate requires either activation of a protein side chain or an organic functional group on the cellulosic substrate. Use of a water soluble carbodiimide to create an amide linkage as the covalent attachment between the enzyme and substrate represents an aqueous-based alternative which may be preferred for textile processes. Here we describe an amide bond-mediated lysozyme immobilization applied to cotton where either the carboxylate side chains of the protein or pendant carboxylates in a citrate, cross-linked cotton support are activated as the O-acyl-isourea intermediate, and the reactive amino nucleophiles are derived from amino-silanized cotton and the protein’s amino side chains, respectively. A comparison is made of the two activation approaches to covalently link lysozyme to two different cotton fabrics using the water soluble carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate. A comparison of the resulting enzyme activities of lysozyme on two different cotton supports showed that linking lysozyme to citrate crosslinked cotton gave higher activity than on aminosilanized cotton. The lysozyme-cellulose conjugate formed on the citrate crosslinked nonwoven cotton fabric gave the highest yield and antimicrobial activity.