Model of Epidemic Kinetics with a Source on the Example of Moscow.

A new theoretical model of epidemic kinetics is considered, which uses elements of the physical model of the kinetics of the atomic level populations of an active laser medium as follows: a description of states and their populations, transition rates between states, an integral operator, and a source of influence. It is shown that to describe a long-term epidemic, it is necessary to use the concept of the source of infection. With a model constant source of infection, the epidemic, in terms of the number of actively infected people, goes to a stationary regime, which does not depend on the population size and the characteristics of quarantine measures. Statistics for Moscow daily increase in infected is used to determine the real source of infection. An interpretation of the waves generated by the source is given. It is shown that more accurate statistics of excess mortality can only be used to clarify the frequency rate of mortality of the epidemic, but not to determine the source of infection.

Prominent Neutralizing Antibody Response Targeting the Ebolavirus Glycoprotein Subunit Interface Elicited by Immunization.

The severe death toll caused by the recent outbreak of Ebola virus disease reinforces the importance of developing ebolavirus prevention and treatment strategies. Here, we have explored the immunogenicity of a novel immunization regimen priming with vesicular stomatitis virus particles bearing Sudan Ebola virus (SUDV) glycoprotein (GP) that consists of GP1 & GP2 subunits and boosting with soluble SUDV GP in macaques, which developed robust neutralizing antibody (nAb) responses following immunizations. Moreover, EB46, a protective nAb isolated from one of the immune macaques, is found to target the GP1/GP2 interface, with GP-binding mode and neutralization mechanism similar to a number of ebolavirus nAbs from human and mouse, indicating that the ebolavirus GP1/GP2 interface is a common immunological target in different species. Importantly, selected immune macaque polyclonal sera showed nAb specificity similar to EB46 at substantial titers, suggesting that the GP1/GP2 interface region is a viable target for ebolavirus vaccine.Importance: The elicitation of sustained neutralizing antibody (nAb) responses against diverse ebolavirus strains remains as a high priority for the vaccine field. The most clinically advanced rVSV-ZEBOV vaccine could elicit moderate nAb responses against only one ebolavirus strain, EBOV, among the five ebolavirus strains, which last less than 6 months. Boost immunization strategies are desirable to effectively recall the rVSV vector-primed nAb responses to prevent infections in prospective epidemics, while an in-depth understanding of the specificity of immunization-elicited nAb responses is essential for improving vaccine performance. Here, using non-human primate animal model, we demonstrated that booster immunization with a stabilized trimeric soluble form of recombinant glycoprotein derived from the ebolavirus Sudan strain following the priming rVSV vector immunization led to robust nAb responses that substantially map to the subunit interface of ebolavirus glycoprotein, a common B cell repertoire target of multiple species including primates and rodents.

Discovery and Preclinical Development of Antigiardiasis Fumagillol Derivatives.

Giardiasis, caused by the intestinal parasite Giardia lamblia, is a severe diarrheal disease, endemic in poverty-stricken regions of the world, and also a common infection in developed countries. The available therapeutic options are associated with adverse effects, and G. lamblia resistance to the standard-of-care drugs is spreading. Fumagillin, an antimicrosporidiosis drug, is a therapeutic agent with potential for the treatment of giardiasis. However, it exhibits considerable, albeit reversible, toxicity when used to treat immunocompromised microsporidiosis patients. Fumagillin is also a highly unstable compound. To address these liabilities, we designed and synthesized stable fumagillol derivatives with lower levels of permeation across polarized epithelial Caco-2 cells and better potency against G. lamblia trophozoites than fumagillin. Metronidazole-resistant G. lamblia strains were also susceptible to the new fumagillol derivatives. In addition, these compounds were more potent against the amebiasis-causing parasite Entamoeba histolytica than fumagillin. Two compounds exhibited better thermal and acid stability than fumagillin, which should prolong the drug shelf life and reduce compound degradation in the stomach. Studies with a mouse model of giardiasis with the most stable compound, 4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-1-yl)oxiran-2-yl)-1-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)benzoic acid (compound 9), revealed that it had better efficacy (effective dose [ED]) than fumagillin at both the fully curative dose (the 100% ED) of 6.6 mg/kg of body weight and a 50% ED of 0.064 mg/kg. Plasma pharmacokinetics revealed the slow absorption of compound 9 through the gut, consistent with the in vitro characterization in Caco-2 cells. An acute-dose study yielded a maximum tolerated dose (MTD) of 1,500 mg/kg, 227-fold higher than the fully curative dose. Thus, along with improved stability, compound 9 also exhibited an excellent therapeutic window.

HIV-1 gp120-CD4-Induced Antibody Complex Elicits CD4 Binding Site-Specific Antibody Response in Mice.

Elicitation of broadly neutralizing Ab (bNAb) responses toward the conserved HIV-1 envelope (Env) CD4 binding site (CD4bs) by vaccination is an important goal for vaccine development and yet to be achieved. The outcome of previous immunogenicity studies suggests that the limited accessibility of the CD4bs and the presence of predominant nonneutralizing determinants (nND) on Env may impede the elicitation of bNAbs and their precursors by vaccination. In this study, we designed a panel of novel immunogens that 1) preferentially expose the CD4bs by selective elimination of glycosylation sites flanking the CD4bs, and 2) minimize the nND immune response by engineering fusion proteins consisting of gp120 Core and one or two CD4-induced (CD4i) mAbs for masking nND epitopes, referred to as gp120-CD4i fusion proteins. As expected, the fusion proteins possess improved antigenicity with retained affinity for VRC01-class, CD4bs-directed bNAbs and dampened affinity for nonneutralizing Abs. We immunized C57BL/6 mice with these fusion proteins and found that overall the fusion proteins elicit more focused CD4bs Ab response than prototypical gp120 Core by serological analysis. Consistently, we found that mice immunized with selected gp120-CD4i fusion proteins have higher frequencies of germinal center-activated B cells and CD4bs-directed memory B cells than those inoculated with parental immunogens. We isolated three mAbs from mice immunized with selected gp120-CD4i fusion proteins and found that their footprints on Env are similar to VRC01-class bNAbs. Thus, using gp120-CD4i fusion proteins with selective glycan deletion as immunogens could focus Ab response toward CD4bs epitope.

Rational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity.

HIV-1 broadly neutralizing antibodies (bNAbs) are being explored as passively administered therapeutic and preventative agents. However, the extensively diversified HIV-1 envelope glycoproteins (Env) rapidly acquire mutations to evade individual bNAbs in monotherapy regimens. The use of a "single" agent to simultaneously target distinct Env epitopes is desirable to overcome viral diversity. Here, we report the use of tandem single-chain variable fragment (ScFv) domains of two bNAbs, specific for the CD4-binding site and V3 glycan patch, to form anti-HIV-1 bispecific ScFvs (Bi-ScFvs). The optimal Bi-ScFv crosslinks adjacent protomers within one HIV-1 Env spike and has greater neutralization breadth than its parental bNAbs. Furthermore, the combination of this Bi-ScFv with a third bNAb recognizing the Env membrane proximal external region (MPER) results in a trispecific bNAb, which has nearly pan-isolate neutralization breadth and high potency. Thus, multispecific antibodies combining functional moieties of bNAbs could achieve outstanding neutralization capacity with augmented avidity.

Discovery of novel antigiardiasis drug candidates.

Giardiasis is a severe intestinal parasitic disease caused by Giardia lamblia, which inflicts many people in poor regions and is the most common parasitic infection in the United States. Current standard care drugs are associated with undesirable side effects, treatment failures, and an increasing incidence of drug resistance. As follow-up to a high-throughput screening of an approved drug library, which identified compounds lethal to G. lamblia trophozoites, we have determined the minimum lethal concentrations of 28 drugs and advanced 10 of them to in vivo studies in mice. The results were compared to treatment with the standard care drug, metronidazole, in order to identify drugs with equal or better anti-Giardia activities. Three drugs, fumagillin, carbadox, and tioxidazole, were identified. These compounds were also potent against metronidazole-resistant human G. lamblia isolates (assemblages A and B), as determined in in vitro assays. Of these three compounds, fumagillin is currently an orphan drug used within the European Union to treat microsporidiosis in immunocompromised individuals, whereas carbadox and tioxidazole are used in veterinary medicine. A dose-dependent study of fumagillin in a giardiasis mouse model revealed that the effective dose of fumagillin was ∼ 100-fold lower than the metronidazole dose. Therefore, fumagillin may be advanced to further studies as an alternative treatment for giardiasis when metronidazole fails.

Structural basis for inactivation of Giardia lamblia carbamate kinase by disulfiram.

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP.

The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK) catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK) with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5'-adenylyl-ß,γ-imidodiphosphate (AMP-PNP), was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123-170), which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent.

A homogenous luminescence assay reveals novel inhibitors for giardia lamblia carbamate kinase.

The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardisis, resistance to these drugs has been reported and is likely to increase. The Giardia carbamate kinase (glCK) plays an essential role in Giardia metabolism and has no homologs in humans, making it an attractive candidate for anti-Giardia drug development. We have developed a luminescent enzyme coupled assay to measure the activity of glCK by quantitating the amount of ATP produced by the enzyme. This assay is homogeneous and has been miniaturized into a 1536-well plate format. A pilot screen against 4,096 known compounds using this assay yielded a signal-to-basal ratio of 11.5 fold and Z' factor of 0.8 with a hit rate of 0.9 % of inhibitors of glCK. Therefore, this Giardia lamblia carbamate kinase assay is useful for high throughput screening of large compound collection for identification of the inhibitors for drug development.

Protein characterization of a candidate mechanism SNP for Crohn's disease: the macrophage stimulating protein R689C substitution.

High throughput genome wide associations studies (GWAS) are now identifying a large number of genome loci related to risk of common human disease. Each such locus presents a challenge in identifying the relevant underlying mechanism. Here we report the experimental characterization of a proposed causal single nucleotide polymorphism (SNP) in a locus related to risk of Crohn's disease and ulcerative colitis. The SNP lies in the MST1 gene encoding Macrophage Stimulating Protein (MSP), and results in an R689C amino acid substitution within the ß-chain of MSP (MSPß). MSP binding to the RON receptor tyrosine kinase activates signaling pathways involved in the inflammatory response. We have purified wild-type and mutant MSPß proteins and compared biochemical and biophysical properties that might impact the MSP/RON signaling pathway. Surface plasmon resonance (SPR) binding studies showed that MSPß R689C affinity to RON is approximately 10-fold lower than that of the wild-type MSPß and differential scanning fluorimetry (DSF) showed that the thermal stability of the mutant MSPß was slightly lower than that of wild-type MSPß, by 1.6 K. The substitution was found not to impair the specific Arg483-Val484 peptide bond cleavage by matriptase-1, required for MSP activation, and mass spectrometry of tryptic fragments of the mutated protein showed that the free thiol introduced by the R689C mutation did not form an aberrant disulfide bond. Together, the studies indicate that the missense SNP impairs MSP function by reducing its affinity to RON and perhaps through a secondary effect on in vivo concentration arising from reduced thermodynamic stability, resulting in down-regulation of the MSP/RON signaling pathway.

Rational design, synthesis and evaluation of first generation inhibitors of the Giardia lamblia fructose-1,6-biphosphate aldolase.

Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn(2+) binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K(i)=14µM) that is comparable to that of FBP (K(m)=2µM) or its inert analog TBP (K(i)=1µM). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3Å) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn(2+). The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn(2+) are not consistent with a strong interaction. To determine if Zn(2+)coordination occurs in the GlFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn(2+) coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn(2+) coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn(2+) coordination to binding of 8 to GlFBPA.

High-throughput Giardia lamblia viability assay using bioluminescent ATP content measurements.

The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardiasis, drug resistance has been reported and is likely to increase, and recurrent infections are common. The search for new drugs that can overcome the drug-resistant strains of Giardia is an unmet medical need. New drug screen methods can facilitate the drug discovery process and aid with the identification of new drug targets. Using a bioluminescent ATP content assay, we have developed a phenotypic drug screen method to identify compounds that act against the actively growing trophozoite stage of the parasite. This assay is homogeneous, robust, and suitable for high-throughput screening of large compound collections. A screen of 4,096 pharmacologically active small molecules and approved drugs revealed 43 compounds with selective anti-Giardia properties, including 32 previously reported and 11 novel anti-Giardia agents. The most potent novel compound was fumagillin, which showed 50% inhibitory concentrations of 10 nM against the WB isolate and 2 nM against the GS isolate.

X-ray structure and characterization of carbamate kinase from the human parasite Giardia lamblia.

Carbamate kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP and ammonium carbamate, which is hydrolyzed to ammonia and carbonate. The three-dimensional structure of carbamate kinase from the human parasite Giardia lamblia (glCK) has been determined at 3 A resolution. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 69.77, b = 85.41, c = 102.1 A, beta = 106.8 degrees . The structure was refined to a final R factor of 0.227. The essentiality of glCK together with its absence in humans makes the enzyme an attractive candidate for anti-Giardia drug development. Steady-state kinetic rate constants have been determined. The k(cat) for ATP formation is 319 +/- 9 s(-1). The K(m) values for carbamoyl phosphate and ADP are 85 +/- 6 and 70 +/- 5 microM, respectively. The structure suggests that three invariant lysine residues (Lys131, Lys216 and Lys278) may be involved in the binding of substrates and phosphoryl transfer. The structure of glCK reveals that a glycerol molecule binds in the likely carbamoyl phosphate-binding site.

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia.

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an insilico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N(alpha)-benzoyl-l-arginine, and N(omega)-amino-l-arginine as substrates but not agmatine, l-homoarginine, N(alpha)-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424-alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k(1)=80s(-1)) and hydrolysis (k(2)=35s(-1)) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k(cat)=2.6s(-1)), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N(omega)-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.

Structural insights into the substrate binding and stereoselectivity of giardia fructose-1,6-bisphosphate aldolase.

Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA) is a member of the class II zinc-dependent aldolase family that catalyzes the cleavage of d-fructose 1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced with an alanine residue renders the enzyme inactive. A comparison of the crystal structures of D83A FBPA in complex with FBP and of wild-type FBPA in the unbound state revealed a substrate-induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn(2+). When FBP binds, the Zn(2+) shifts up to 4.6 A toward the catalytic Asp83, which brings the metal within coordination distance of the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor d-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analogue of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA-FBP structure. The mechanism invokes coordination and coplanarity of the Zn(2+) with the FBP's O-C(3)-C(4)-O group concomitant with coordination of the Asp83 carboxylic group. Catalysis is accompanied by movement of Zn(2+) to a site coplanar with the O-C(2)-C(3)-O group of the DHAP. glFBPA exhibits strict substrate specificity toward FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP versus TBP specificity. Replacement of Asp255 with an alanine resulted in an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; k(cat)/K(m) = 80 M(-1) s(-1)) while maintaining activity toward FBP at a 50-fold lower catalytic efficacy compared with that of wild-type FBPA. The collection of structures and sequence analyses highlighted additional residues that may be involved in substrate discrimination.

Inactivation of microbial arginine deiminases by L-canavanine.

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.

Characterization, kinetics, and crystal structures of fructose-1,6-bisphosphate aldolase from the human parasite, Giardia lamblia.

Class I and class II fructose-1,6-bisphosphate aldolases (FBPA), glycolytic pathway enzymes, exhibit no amino acid sequence homology and utilize two different catalytic mechanisms. The mammalian class I FBPA employs a Schiff base mechanism, whereas the human parasitic protozoan Giardia lamblia class II FBPA is a zinc-dependent enzyme. In this study, we have explored the potential exploitation of the Giardia FBPA as a drug target. First, synthesis of FBPA was demonstrated in Giardia trophozoites by using an antibody-based fluorescence assay. Second, inhibition of FBPA gene transcription in Giardia trophozoites suggested that the enzyme is necessary for the survival of the organism under optimal laboratory growth conditions. Third, two crystal structures of FBPA in complex with the transition state analog phosphoglycolohydroxamate (PGH) show that the enzyme is homodimeric and that its active site contains a zinc ion. In one crystal form, each subunit contains PGH, which is coordinated to the zinc ion through the hydroxamic acid hydroxyl and carbonyl oxygen atoms. The second crystal form contains PGH only in one subunit and the active site of the second subunit is unoccupied. Inspection of the two states of the enzyme revealed that it undergoes a conformational transition upon ligand binding. The enzyme cleaves d-fructose-1,6-bisphosphate but not d-tagatose-1,6-bisphosphate, which is a tight binding competitive inhibitor. The essential role of the active site residue Asp-83 in catalysis was demonstrated by amino acid replacement. Determinants of catalysis and substrate recognition, derived from comparison of the G. lamblia FBPA structure with Escherichia coli FBPA and with a closely related enzyme, E. coli tagatose-1,6-bisphosphate aldolase (TBPA), are described.

Kinetic analysis of Pseudomonas aeruginosa arginine deiminase mutants and alternate substrates provides insight into structural determinants of function.

L-Arginine deiminase from Pseudomonas aeruginosa (PaADI) catalyzes the hydrolysis of arginine to citrulline and ammonia. PaADI belongs to the guanidino group-modifying enzyme superfamily (GMSF), which conserves backbone fold and a Cys-, His-, and Asp-based catalytic core. In this paper the contributions made by the PaADI core residues Cys406, His278, and Asp166 and the contribution from the neighboring Asp280 (conserved in most but not all GMSF members) to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were accessed by kinetic analysis of site-directed mutants. In addition, solution hydrolysis in a chemical model of the S-alkylthiouronium intermediate was examined to reveal the importance of general base catalysis in the enzymatic reaction. Substitutions of the active site gating residue Arg401, the l-arginine C(alpha)NH(3)(+)(COO(-)) binding residues, Arg185, Arg243, and Asn160, or the His278 hydrogen bond partner, Glu224, were found to cause dramatic reductions in the enzyme turnover rate. These results are interpreted to suggest that electrostatic interactions play a dominant role in PaADI catalysis. Structural variations observed in P. aeruginosa GMSF enzymes PaADI, agmatine deiminase (PaAgDI), and N(omega),N(omega)-dimethylarginine dimethylaminohydrolase (PaDDAH) indicate an early divergence of the encoding genes. Arginine analogues that are known substrates for PaAgDI and PaDDAH were tested with PaADI to define clear boundaries of biochemical function in the three hydrolases. The conservation of a catalytic core associated with the common chemical function and the divergence of substrate-binding residues (as well as one key catalytic residue) to expand the substrate range provide insight into the evolution of the catalysts that form the GMSF.

DnaK from Vibrio proteolyticus: complementation of a dnaK-null mutant of Escherichia coli and the role of its ATPase domain.

We cloned the 4.8-kbp DNA fragment containing the dnaK gene from the chromosomal DNA of Vibrio proteolyticus. It contained four genes arranged unidirectionally in the order of grpE, gltP, dnaK and dnaJ. The DnaK gene of V. proteolyticus (VprDnaK) allowed a dnaK-null mutant of Escherichia coli (DeltadnaK52) to propagate lambda phages but not to grow at 43 degrees C. However, a chimeric DnaK gene comprising the regions corresponding to the N-terminal ATPase domain of E. coli DnaK (EcoDnaK) and the C-terminal region of VprDnaK including the substrate-binding domain, enabled the mutant to grow at 43 degrees C. The temperature dependence for the ATPase activity of the chimeric DnaK was similar to that of EcoDnaK. Fluorometric analyses showed that the chimeric DnaK is much more thermostable than EcoDnaK and VprDnaK. These findings indicate that the thermal stability of the ATPase domain of DnaK is responsible for its chaperone action at high temperatures such as 43 degrees C.