Identification and characterization of a critical region in the glycogen synthase from Escherichia coli

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.     

The amino acidic substitution of cysteine 167 by serine (C167S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability.

The restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation C134S was purified to homogeneity but appeared to be very unstable. In contrast, the C167S mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the C167S BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease.     

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.     

Soluble human core 2 beta6-N-acetylglucosaminyltransferase C2GnT1 requires its conserved cysteine residues for full activity

Human UDP-GlcNAc: Galbeta1-3GalNAc- (GlcNAc to GalNAc) beta1,6-GlcNAc-transferase (C2GnT1) is a member of a group of beta6-GlcNAc-transferases that belongs to CAZy family 14. One of the striking features of these beta6-GlcNAc-transferases is the occurrence of nine completely conserved cysteine residues that are located throughout the catalytic domain. We have expressed the soluble catalytic domain of human C2GnT1 in insect cells, and isolated active enzyme as a secreted protein. beta-Mercaptoethanol (beta-ME) and dithiothreitol (DTT) were found to stimulate the enzyme activity up to 20-fold, indicating a requirement for a reduced sulfhydryl for activity. When the enzyme was subjected to nonreducing PAGE, the migration of the protein was identical to the migration in reducing gels, demonstrating the absence of intermolecular disulfide bonds. This suggested that the monomer is the active form of the enzyme. Sulfhydryl reagents such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) inactivated the enzyme, and the inactivation was partially prevented by prior addition of donor or acceptor substrate and by sulfhydryl reducing agents. We therefore investigated the role of all nine conserved cysteine residues in enzyme stability and activity by site-directed mutagenesis where individual cysteine residues were changed to serine. All of the mutants were expressed as soluble proteins. Seven of the Cys mutants were found to be inactive, while C100S and C217S mutants had 10% and 41% activity, respectively, when compared to the wild-type enzyme. Wild-type and C217S enzymes had similar K(M) and V(max) values for acceptor substrate Galbeta1-3GalNAcalpha-p-nitrophenyl (GGApnp), but the K(M) value for UDP-GlcNAc was higher for C217S than for the wild-type enzyme. In contrast to wild-type enzyme, C217S was not stimulated by reducing agents and was not inhibited by sulfhydryl specific reagents. These results suggest that Cys-217 is a free sulfhydryl in active wild-type enzyme and that Cys-217, although not required for activity, is in or near the active site of the protein. Since seven of the mutations were totally inactive, it is likely that these seven Cys residues play a role in maintaining an active conformation of soluble C2GnT1 by forming disulfide bonds. These bonds are only broken at high concentrations of disulfide reducing agents.     

Key residues of loop 3 in the interaction with the interface residue at position 14 in triosephosphate isomerase from Trypanosoma brucei

Cysteine 14 is an interface residue that is fundamental for the catalysis and stability of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM). Its side chain is surrounded by a deep pocket of 11 residues that are part of loop 3 of the adjacent monomer. Mutation of this residue to serine (producing single mutant C14S) yields a wild-type-like enzyme that is resistant to the action of sulfhydryl reagents methylmethane thiosulfonate (MMTS) and 5,5-dithiobis(2-nitrobenzoate) (DTNB). This mutant enzyme was a starting point for probing by cysteine scanning the role of four residues of loop 3 in the catalysis and stability of the enzyme. Considering that the conservative substitution of either serine or alanine with cysteine would minimally alter the structure and properties of the environment of the residue in position 14, we made double mutants C14S/A69C, C14S/S71C, C14S/A73C, and C14S/S79C. Three of these double mutants were similar in their kinetic parameters to wild-type TbTIM and the single mutant C14S, but double mutant C14S/A73C showed a greatly reduced k cat. All enzymes had similar CD spectra, but all mutants had thermal stabilities lower than that of wild-type TbTIM. Intrinsic fluorescence was also similar for all enzymes, but the double mutants bound up to 50 times more 1-anilino-8-naphthalene sulfonate (ANS) and were susceptible to digestion with subtilisin. The double mutants were also susceptible to inactivation by sulfhydryl reagents. Double mutant C14S/S79C exhibited the highest sensitivity to MMTS and DTNB, bound a significant amount of ANS, and had the highest sensitivity to subtilisin. Thus, the residues at positions 73 and 79 are critical for the catalysis and stability of TbTIM, respectively.     

Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E. coli) contains four cysteine residues. In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis. Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside. Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity. These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis. However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E. coli ArgRS were not essential to the enzymatic activity. Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320). Our study suggested that inactivation of E. coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.     

Thermodynamic and kinetic destabilization of triosephosphate isomerase resulting from the mutation of conserved and non-conserved cysteines

Several variants of Saccharomyces cerevisiae triosephosphate isomerase (yTIM) were studied to determine how mutations of conserved and non-conserved Cys residues affect the enzyme. Wild-type yTIM has two buried free cysteines: Cys 41 (non-conserved) and the invariant Cys 126. Single-site mutants, containing substitutions of these cysteines with Ala, Val, or Ser (the three most conservative changes for a buried Cys, according to substitution matrices), were examined for stability and enzymatic activity. Neither of the Cys residues was found to be essential for enzyme catalysis. Determination of the global stability of the mutants indicated that, regardless of which Cys was substituted, individual Cys→Ala and Cys→Val mutations, as well as the C41S substitution, all decrease the unfolding free energy of the dimeric protein by less than 23 kJ mol(-1) (at 37 °C, pH 7.4), as compared to the wild-type enzyme. In contrast, a substantially larger destabilization (37 kJ mol(-1)) was found in the C126S mutant. These results suggest that, with the exception of C126S, all of these mutations can be regarded as neutral (i.e., mutations that do not impair the reproductive success of the organism). Accordingly, Cys 126 has remained invariant across evolution because its neutral substitutions by Ala or Val would require a highly unlikely, concerted double mutation at any of the Cys codons. Furthermore, detrimental effects to a cell expressing the C126S TIM mutant more likely arise from the high unfolding rate of this enzyme.     

Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A beta-lactamase Toho-1

The production of class A beta-lactamases is a major cause of clinical resistance to beta-lactam antibiotics. Some of class A beta-lactamases are known to have a disulfide bridge. Both narrow spectrum and extended spectrum beta-lactamases of TEM and the SHV enzymes possess a disulfide bond between Cys77 and Cys123, and the enzymes with carbapenem-hydrolyzing activity have a well-conserved disulfide bridge between Cys69 and Cys238. We produced A77C/G123C mutant of the extended-spectrum beta-lactamase Toho-1 in order to introduce a disulfide bond between the cysteine residues at positions 77 and 123. The result of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) titrations confirmed formation of a new disulfide bridge in the mutant. The results of irreversible heat inactivation and circular dichroism (CD) melting experiments indicated that the disulfide bridge stabilized the enzyme significantly. Though kinetic analysis indicated that the catalytic properties of the mutant were quite similar to those of the wild-type enzyme, E. coli producing this mutant showed drug resistance significantly higher than E. coli producing the wild-type enzyme. We speculate that the stability of the enzymes provided by the disulfide bond may explain the wide distribution of TEM and SHV derivatives and explain how various mutations can cause broadened substrate specificity without loss of stability.     

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.     

Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1).

Biosynthesis of mucin-type O-glycans is initiated by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine-specific reagent, p-chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc-T1). Presence of either UDP-GalNAc or UDP during PCMPS treatment protected GalNAc-T1 from inactivation, to the same extent. This suggests that GalNAc-T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc-T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc-T1, as free cysteine residues, by cysteine-specific labeling of GalNAc-T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine-mutants for UDP-GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP-GalNAc.     

Escherichia coli cyclopropane fatty acid synthase: is a bound bicarbonate ion the active-site base?

Cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acid using S-adenosyl-L-methionine as the methylene donor. The crystal structure of three cyclopropane synthases from Mycobacterium tuberculosis showed a bicarbonate ion bound in the active site that was proposed to act as a general base in the reaction mechanism [Huang, C., Smith, V., Glickman, M. S., Jacobs, W. R., and Sacchettini, J. C. (2002) J. Biol. Chem. 277, 11559-11569]. Because the in vitro activity of M. tuberculosis cyclopropane synthases has not yet been reported and because the ligands of the bicarbonate ion are all strictly conserved in cyclopropane synthases, we used the closely related Escherichia coli cyclopropane fatty acid synthase for this study. The putative ligands that share a hydrogen bond with the bicarbonate through their side chains were mutated. H266A, Y317F, E239A, and E239Q mutants were thus constructed and purified, and their catalytic efficiencies were 5.3, 0.7, 0.2, and <0.02%, respectively. C139 that is bound to the bicarbonate by its NH amide had already been mutated to serine in a previous work, and this mutant retains 31% of the activity of the wild-type enzyme. Kinetic analyses and binding studies using spectrofluorimetry showed that these mutations affected the catalytic constant rather than the binding of the substrates. While addition of free bicarbonate had almost no effect on the wild-type enzyme activity, all mutants, with the exception of E239A and E239Q, were rescued by the addition of free bicarbonate. The catalytic efficiencies of the rescued mutants were 85, 16, and 14% for C139S, H266A, and Y317F, respectively. This effect was specific to bicarbonate. The kinetic parameters of the rescued mutants were determined, and it is shown that the rescuing effect is due to an increase in kcat. These data are interpreted by assuming that the E. coli cyclopropane fatty acid synthase specifically binds a bicarbonate ion that is involved in catalysis, as proposed for the M. tuberculosis enzymes, and that mutation of the bicarbonate ligands decreases the affinity for that ion. However, because the E239Q mutation could not be rescued, we propose that E239 forms a catalytic dyad with the bicarbonate to perform the proton abstraction necessary in the chemical pathway to the cyclopropane ring.     

Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide

Pig heart NADP-specific isocitrate dehydrogenase is inactivated by N-ethylmaleimide (NEM) (Colman, R. F., and Chu, R. (1970) J. Biol. Chem. 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+. We have now treated the enzyme with [3H]NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides. In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CATITPDEAR and EPIICK, were labeled at Cys. Upon reaction with [3H]NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis. The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM. The Cys in SSGGFVWACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate. Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with [14C]iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced. Six corresponding peptides were isolated from enzyme treated under denaturing conditions with [3H]NEM. These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme. A comparison of the sequences of the NH2-terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.     

Identification of new inhibitors of E. coli cyclopropane fatty acid synthase using a colorimetric assay

Bacterial cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acids by transferring a methylene group from S-adenosyl-L-methionine (AdoMet) to the double bond of the lipids. Mycobacterium tuberculosis cyclopropane synthases have been shown to be implicated in pathogenicity, and therefore constitute attractive targets for the development of new drugs against tuberculosis. However, no in vitro assay for these cyclopropane synthases has yet been described. The homologous E. coli enzyme, cyclopropane fatty acid synthase, is thus a valuable model for inhibitor screening. Here, we report the adaptation to the E. coli CFAS of a previously reported enzyme-coupled colorimetric assay based on the quantification, using Ellman's reagent, of homocysteine produced from S-adenosyl-L-homocysteine, a product of the reaction, in the presence of AdoHcy nucleosidase and S-ribosylhomocysteinase. Using this assay we measured the kinetic parameters for CFAS: Km (AdoMet)=80 microM, kcat=4 min(-1). We adapted this assay to microtiter plates and tested 15 potential inhibitors of CFAS. Among them, two new inhibitors, a lipid analog and a thioether analog of AdoHcy, showed IC50 of 4 microM and 11 microM, respectively. This new assay will thus be useful for high-throughput screening of compound libraries for discovering novel antituberculous drug candidates.     

Identification of the reactive cysteines of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis

Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.     

A mutagenic study of beta-1,4-galactosyltransferases from Neisseria meningitidis

N-terminal His-tagged recombinant beta-1,4-galactosyltransferase from Neisseria meningitidis was expressed and purified to homogeneity by column chromatography using Ni-NTA resin. Mutations were introduced to investigate the roles of, Ser68, His69, Glu88, Asp90, and Tyr156, which are components of a highly conserved region in recombinant beta-1,4 galactosyltransferase. Also, the functions of three other cysteine residues, Cys65, Cys139, and Cys205, were investigated using site-directed mutagenesis to determine the location of the disulfide bond and the role of the sulfhydryl groups. Purified mutant galactosyltransferases, His69Phe, Glu88Gln and Asp90Asn completely shut down wild-type galactosyltransferase activity (1-3 %). Also, Ser68Ala showed much lower activity than wild-type galactosyltransferase (19 %). However, only the substitution of Tyr156Phe resulted in a slight reduction in galactosyltransferase activity (90 %). The enzyme was found to remain active when the cysteine residues at positions 139 and 205 were replaced separately with serine. However, enzyme reactivity was found to be markedly reduced when Cys65 was replaced with serine (27 %). These results indicate that conserved amino acids such as Cys65, Ser68, His69, Glu88, and Asp90 may be involved in the binding of substrates or in the catalysis of the galactosyltransferase reaction.     

A peptide substrate-based affinity label blocks protein kinase C-catalyzed ATP hydrolysis and peptide-substrate phosphorylation.

Studies focused on the cAMP-dependent protein kinase (PKA) have led to the identification of conserved active-site residues involved in Ser/Thr protein kinase catalysis and have ruled out a role for Cys residues in the catalytic mechanism. Protein kinase C (PKC) is a Ser/Thr protein kinase isozyme family. We recently reported that the peptide-substrate analog N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu (N-biotinyl-RRRCLRRL) spontaneously forms intermolecular disulfide bridges with the active-site region of PKC isozymes concomitant with inactivation of histone kinase catalysis. Because Cys does not participate in PKC catalysis, one can analyze the active-site topology of PKC by examining which catalytic reactions are sterically hindered when the inactivator peptide is tethered to Cys in the active-site region of the enzyme. In this report, we show that N-biotinyl-RRRCLRRL inactivates the bulky PKC-catalyzed histone phosphorylation reaction, the comparatively less bulky PKC-catalyzed phosphorylation of a series of octapeptide, hexapeptide, and pentapeptide substrates, the intramolecular autophosphorylation reaction of PKC, and the least bulky PKC-catalyzed reaction, ATP hydrolysis, in a dithiothreitol-sensitive manner with comparable efficacy. Our results provide evidence that the covalent linkage of N-biotinyl-RRRCLRRL to the active-site region of PKC sterically hinders PKC catalysis, even in the absence of peptide and protein substrates.     

Identification of inhibitors of the E. coli cyclopropane fatty acid synthase from the screening of a chemical library: In vitro and in vivo studies

Using an automated coupled colorimetric assay for the Escherichia coli cyclopropane fatty acid synthase (CFAS), we have screened an academic chemical library of 3040 compounds, to identify new inhibitors of this enzyme. We identified 8 compounds as potent inhibitors of this enzyme, with IC(50) ranging from 1 to 10 microM, in the presence of 750 microM S-adenosyl-l-methionine and 1 mg/mL phospholipids. We conducted kinetic analyses of the inhibition of the CFAS using dioctylamine and three inhibitors identified in this report: sinefungin, 1, a synthetic S-adenosyl-l-homocysteine analog, 2, and an indoloquinolizine derivative, 3. The inhibition patterns observed were interpreted assuming that the E. coli CFAS operated via an ordered Bi Bi mechanism with binding of S-adenosyl-l-methionine first. Dioctylamine was the most potent inhibitor with a competitive inhibition constant of 130 nM with respect to the phospholipids. Compound 2 bound to the two substrate-binding sites of the enzyme suggesting that it acted as a bisubstrate analog (apparent inhibition constant, K(I)=6 microM). Compound 2 was also found to completely inhibit cyclopropanation of the phospholipids in growing E. coli cells, at 150 microM. This molecule is thus the first inhibitor of a cyclopropane synthase that is active in vivo, contrary to sinefungin and other analogs that are only active on the isolated enzyme.     

Oxygen-independent coproporphyrinogen-III oxidase HemN from Escherichia coli

In bacteria the oxygen-independent coproporphyrinogen-III oxidase catalyzes the oxygen-independent conversion of coproporphyrinogen-III to protoporphyrinogen-IX. The Escherichia coli hemN gene encoding a putative part of this enzyme was overexpressed in E. coli. Anaerobically purified HemN is a monomeric protein with a native M(r) = 52,000 +/- 5,000. A newly established anaerobic enzyme assay was used to demonstrate for the first time in vitro coproporphyrinogen-III oxidase activity for recombinant purified HemN. The enzyme requires S-adenosyl-l-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis. An oxygen-sensitive iron-sulfur cluster was identified by absorption spectroscopy and iron analysis. Cysteine residues Cys(62), Cys(66), and Cys(69), which are part of the conserved CXXXCXXC motif found in all HemN proteins, are essential for iron-sulfur cluster formation and enzyme function. Completely conserved residues Tyr(56) and His(58), localized closely to the cysteine-rich motif, were found to be important for iron-sulfur cluster integrity. Mutation of Gly(111) and Gly(113), which are part of the potential GGGTP S-adenosyl-l-methionine binding motif, completely abolished enzymatic function. Observed functional properties in combination with a recently published computer-based enzyme classification (Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001) Nucleic Acids Res. 29, 1097-1106) identifies HemN as "Radical SAM enzyme." An appropriate enzymatic mechanism is suggested.     

Molecular discrimination of type-I over type-II methionyl aminopeptidases

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.