The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 A resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.
Mechanistic studies have been carried out on the bacterial enzyme UDP-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This enzyme is interesting because it epimerizes a stereocenter that does not bear an acidic proton, and therefore it cannot utilize a simple deprotonation/reprotonation mechanism. A coupled enzyme assay employing UDP-ManNAc dehydrogenase has been developed. The epimerization in D2O is found to be accompanied by the incorporation of deuterium into the C-2‘‘ position of both epimers, supporting a mechanism that ultimately involves a proton transfer at this position. The epimerization of [2‘‘-2H]UDP-GlcNAc is slowed by a primary kinetic isotope effect indicating that C−H bond cleavage is occurring during a rate-determining step of the reaction. A positional isotope exchange (PIX) experiment shows that an 18O label in the sugar-UDP bridging position will scramble into nonbridging diphosphate positions during enzymatic epimerization. These observations are consistent with a mechanism that proceeds via cleavage of the anomeric C−O bond, with 2-acetamidoglucal and UDP as enzyme-bound intermediates. Additional evidence for this mechanism is found in the unusual observation that during extended incubations, the intermediates are gradually released from the enzyme and accumulate in solution. These intermediates are formed by an anti elimination of UDP from UDP-GlcNAc and a syn elimination of UDP from UDP-ManNAc. It is likely that E1-like eliminations via oxocarbenium intermediates are involved in the reaction. Further experiments show that 3‘‘-deoxy-UDP-GlcNAc is not a substrate for the enzyme and that the enzyme does not contain a tightly bound NAD+ cofactor.
UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD ؉ -dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDPglucuronate is released last. UDP-xylose was found to be a competitive inhibitor (K i , 2.7 M) of the enzyme. The enzyme is irreversibly inactivated by uridine 5 -diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (k i /K i ) was found to be 2 ؋ 10 3 mM ؊1 min ؊1 . Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5 -diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.The enzyme UDP-glucose dehydrogenase (UDPGDH) 1 catalyzes the NAD ϩ -dependent oxidation of UDP-glucose to UDPglucuronic acid (Fig. 1). It belongs to a small group of dehydrogenases that are able to carry out the 2-fold oxidation of an alcohol to an acid without the release of an aldehyde intermediate (1). Much of the work to date has focused on the properties and mechanism of the beef liver enzyme, and relatively little is known about the enzyme purified from bacterial sources (2-4). In many strains of bacteria that act as human pathogens, UDPGDH provides the UDP-glucuronic acid required for the construction of an antiphagocytic capsular polysaccharide. It is well established that the formation of the capsule is required for virulence (5, 6), and it is thought that the capsule enables the bacteria to evade the host's immune system (7,8). Group A and C streptococci are mammalian pathogens that use UDPGDH in the synthesis of a capsule composed of hyaluronic acid (a polysaccharide consisting of alternating glucuronic acid and N-acetylglucosamine residues) (9, 10). Many of the known strains of Streptococcus pneumoniae also use UDP-glucuronic acid in the construction of their polysaccharide capsule (11), and it has recently been shown that UDPGDH is required for capsule production in S. pneumoniae type 3 (12). The encapsulated Escherichia coli K5 is also known to use the enzyme for a similar purpose (2, 13). The hasB gene that encodes for UDPGDH in group A streptococci (Streptococcus pyogenes) has been cloned and overexpressed in Escherichia coli (14). Its gene product shares 57% sequence identity and 74% sequence similarity with the S. pneumoniae enzyme (15, 16). These properties make th...
The enzyme UDP-N-acetylglucosamine 2-epimerase catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc) in both Grampositive and Gram-negative bacteria (Figure 1). 1 This provides the bacteria with a source of activated ManNAc residues for use in the biosynthesis of cell wall surface polysaccharides. 2 This epimerase is unlike most known racemases and epimerases in that it must invert a stereogenic center that is not adjacent to an electron-withdrawing carbonyl or carboxylate group and therefore cannot employ a simple deprotonation-reprotonation reaction mechanism. 3 A previous report suggested that the enzyme overcomes this obstacle by transiently oxidizing the C-3 hydroxyl of the GlcNAc residue to a ketone, thus acidifying the proton at C-2. 4,5 Deprotonation at C-2, followed by reprotonation on the opposite face and finally reduction of the ketone, would produce the epimeric sugar nucleotide. In this communication we report evidence in favor of an alternative mechanism that proceeds via cleavage of the anomeric C-O bond, with 2-acetamidoglucal and UDP as enzyme-bound intermediates (Figure 1). 6 We have employed a positional isotope exchange (PIX) experiment 7 in which an 18 O label in the sugar-UDP bridging position (darkened atom in Figure 1) has been observed to scramble into nonbridging diphosphate positions during enzymatic epimerization. We have also demonstrated that the enzyme occasionally releases these relatively stable intermediates into solution.The gene coding for the Escherichia coli UDP-GlcNAc 2-epimerase, known as rffE, had tentatively been assigned to an open reading frame, o355, near min 85 on the E. coli chromosome. 8 We have found that rffE is actually located 2.4 kb upstream of this sequence and has been designated nfrC in other work. 9 The nfrC gene product has been overexpressed and was reported to be a cytoplasmic protein of unknown activity that is required for bacteriophage N4 adsorption. 10 We have purified this protein to homogeneity and demonstrated that it is UDP-GlcNAc 2-epimerase. 11
COVER PICTUREThe cover picture shows a ligand-targeted proteinase enzyme or ™catalytic antagonist∫ acting as a molecular angler fish: By precisely positioning different binding ligands (L) around the active site ™mouth∫ of a degradative proteinase enzyme, target proteins (TP) can be plucked from solution, locked in position adjacent to the catalytic triad ™jaws∫, and in this way readily and specifically degraded. The hunting strategy of the deep sea angler fish, which uses a lure above its mouth, illustrates this principle. Further details can be found in the article by B. Davis, R. R. Bott, J. B. Jones et. al. on pp. 533 ± 537. REVIEWS Don't get stressed! Stress is often linked by anecdote to degeneration and disease. Key molecular, cellular and medical evidence is described in this review that gives firm support for this linkage and suggests that key stress hormones such as cortisol or corticosterone acting in synergy with the action of cytokines (see figure) may trigger and/or promote many diseases.N. Lozovaya, A. D. Miller* ± 484Chemical Neuroimmunology: Health in a NutshellChemBioChem is a European journal of chemical biology; it is co-owned by a group of European scientific societies and published by WILEY-VCH. Contributions in ChemBioChem cover chemical biology and biological chemistry, medicinal chemistry, bioinorganic and bioorganic chemistry, biochemistry, molecular and structural biology, that is, research of the overlapping areas between biology and chemistry.
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