Targeting the fatty acid biosynthesis enzyme, beta-ketoacyl-acyl carrier protein synthase III (PfKASIII), in the identification of novel antimalarial agents.

The importance of fatty acids to the human malaria parasite, Plasmodium falciparum, and differences due to a type I fatty acid synthesis (FAS) pathway in the parasite, make it an attractive drug target. In the present study, we developed and a utilized a pharmacophore to select compounds for testing against PfKASIII, the initiating enzyme of FAS. This effort identified several PfKASIII inhibitors that grouped into various chemical classes of sulfides, sulfonamides, and sulfonyls. Approximately 60% of the submicromolar inhibitors of PfKASIII inhibited in vitro growth of the malaria parasite. These compounds inhibited both drug sensitive and resistant parasites and testing against a mammalian cell line revealed an encouraging in vitro therapeutic index for the most active compounds. Docking studies into the active site of PfKASIII suggest a potential binding mode that exploits amino acid residues at the mouth of the substrate tunnel.

Application of 3D-QSAR for identification of descriptors defining bioactivity of antimicrobial peptides.

In our laboratory, a series of antimicrobial peptides have been developed, where the resulting 3D-physicochemical properties are controlled by the placement of amino acids with well-defined properties (hydrophobicity, charge density, electrostatic potential, and so on) at specific locations along the peptide backbone. These peptides exhibited different in vitro activity against Staphylococcus aureus (SA) and Mycobacterium ranae (MR) bacteria. We hypothesized that the differences in the biological activity is a direct manifestation of different physicochemical interactions that occur between the peptides and the cell membranes of the bacteria. 3D-QSAR analysis has shown that, within this series, specific physicochemical properties are responsible for antibacterial activity and selectivity. There are five physicochemical properties specific to the SA QSAR model, while five properties are specific to the MR QSAR model. These results support the hypothesis that, for any particular AMP, organism selectivity and potency are controlled by the chemical composition of the target cell membrane.

Identification of catalytic cysteine, histidine, and lysine residues in Escherichia coli homoserine transsuccinylase.

Homoserine transsuccinylase catalyzes the succinylation of homoserine in several bacterial species, the first unique step in methionine biosynthesis in these organisms. The enzyme from Escherichia coli is reported to be a dimer and uses a ping-pong catalytic mechanism involving transfer of succinate from succinyl-CoA to an enzyme nucleophile, followed by transfer to homoserine to form O-succinylhomoserine. Site-directed mutagenesis and steady-state kinetics were used to identify three amino acids that participate in catalysis. Mutation of cysteine-142 to serine or alanine eliminated all measurable activity, suggesting this amino acid acts as the catalytic nucleophile. Cysteine nucleophiles are often deprotonated by histidine residues, and histidine-235 was identified as the sole absolutely conserved histidine residue among family members. This residue was mutated to both alanine and asparagine, and no activity was observed with either mutant. Lysine-47 had been previously identified as an essential residue. Mutation of this amino acid to arginine reduced catalytic activity by greater than 90%, while mutation to alanine yielded an enzyme with <1% of wild-type activity. A pH-rate profile of the K47R mutant demonstrated that this amino acid participates in the first half reaction. The data presented here provide the first detailed description of the homoserine transsuccinylase active site and provide a framework for additional mechanistic characterization of this enzyme.

Synthesis and biological evaluation of thiazolidine-2-one 1,1-dioxide as inhibitors of Escherichia coli beta-ketoacyl-ACP-synthase III (FabH).

A series of cyclic sulfones has been synthesized and their activity against beta-ketoacyl-ACP-synthase III (FabH) has been investigated. The compounds are selectively active against Escherichia coli FabH (ecFabH), but not Mycobacterium tuberculosis FabH (mtFabH) or Plasmodium falciparum KASIII (PfKASIII). The activity against ecFabH ranges from 0.9 to >100microM and follows a consistent general SAR trend. Many of the compounds were shown to have antimalarial activity against chloroquine (CQ)-sensitive (D6) P. falciparum (IC(50)=5.3microM for the most potent inhibitor) and some were active against E. coli (MIC=6.6microg/ml for the most potent inhibitor).

Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase.

B(12)-dependent methionine synthase (MetH) is a large modular enzyme that utilizes the cobalamin cofactor as a methyl donor or acceptor in three separate reactions. Each methyl transfer occurs at a different substrate-binding domain and requires a different arrangement of modules. In the catalytic cycle, the cobalamin-binding domain carries methylcobalamin to the homocysteine (Hcy) domain to form methionine and returns cob(I)alamin to the folate (Fol) domain for remethylation by methyltetrahydrofolate (CH(3)-H(4)folate). Here, we describe crystal structures of a fragment of MetH from Thermotoga maritima comprising the domains that bind Hcy and CH(3)-H(4)folate. These substrate-binding domains are (beta alpha)(8) barrels packed tightly against one another with their barrel axes perpendicular. The properties of the domain interface suggest that the two barrels remain associated during catalysis. The Hcy and CH(3)-H(4)folate substrates are bound at the C termini of their respective barrels in orientations that position them for reaction with cobalamin, but the two active sites are separated by approximately 50 A. To complete the catalytic cycle, the cobalamin-binding domain must travel back and forth between these distant active sites.

Betaine-homocysteine methyltransferase: zinc in a distorted barrel.

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.

Domain alternation switches B(12)-dependent methionine synthase to the activation conformation.

B(12)-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that uses bound cobalamin as an intermediate methyl carrier. Major domain rearrangements have been postulated to explain how cobalamin reacts with three different substrates: homocysteine, methyltetrahydrofolate and S-adenosylmethionine (AdoMet). Here we describe the 3.0 A structure of a 65 kDa C-terminal fragment of MetH that spans the cobalamin- and AdoMet-binding domains, arranged in a conformation suitable for the methyl transfer from AdoMet to cobalamin that occurs during activation. In the conversion to the activation conformation, a helical domain that capped the cofactor moves 26 A and rotates by 63 degrees, allowing formation of a new interface between cobalamin and the AdoMet-binding (activation) domain. Interactions with the MetH activation domain drive the cobalamin away from its binding domain in a way that requires dissociation of the axial cobalt ligand and, thereby, provide a mechanism for control of the distribution of enzyme conformations.