Escherichia coli 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH).These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9 -91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues. 5Ј-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN)1 functions at two steps in bacterial pathways related to polyamine biosynthesis, quorum sensing, methylation, and purine and methionine salvage reactions ( Fig. 1; see Refs. 1-4). It catalyzes the physiologically irreversible hydrolysis of 5Ј-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose (MTR). The product adenine is subsequently recycled into the adenine nucleotide pool by the widely distributed adenine phosphoribosyltransferase (5), and the 5-methylthio-D-ribose is subsequently phosphorylated to 5-methylthio-␣-D-ribose 1-phosphate and converted into methionine (6). MTA is a by-product of the reactions involving sequential transfers of the aminopropyl group from decarboxylated S-adenosylmethionine to form spermidine and spermine (although spermine is absent in Escherichia coli). Polyamine synthesis is sensitive to product inhibition by MTA, with inhibition constants reported to be 0.3 M for bovine spermine synthase (7) and 50 M for rat spermidine synthase (8). Inhibition of MTAN is therefore expected to inhibit polyamine biosynthesis and the salvage pathways for adenine and methionine. Another function of MTAN in bacteria is generation of S-ribosylhomocysteine (SRH) from SAH. SRH is a precursor for synthesis of tetrahydrofurans, quorum-sensing molecules involved in expression of the enzymes for biofilm formation, exotoxin synthesis, and antibiotic resistance factors (9 -14). Previously characterized nucleoside and nucleotide N-ribosyl hydrolases proceed through transition state structures in which the N-ribosidic

Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine synthesis are recycled in a novel pathway in which 5 -methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5 -methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5 -methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5 -Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.

SummaryBacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP) Malaria is caused by the protozoan parasites Plasmodium falciparum, ovale, vivax, and malariae and is responsible for an estimated 1-2 million deaths per year (1, 2). Controlling this disease with current anti-malarials has become more difficult because of emerging drug-resistant strains (1, 2). Therefore, the validation of alternative anti-malarial targets is crucial to development of new chemotherapeutic approaches. The purine salvage pathway has been identified as a potential anti-malarial target (3, 4). Unlike its host, Plasmodium cannot synthesize purines de novo and depends exclusively on purine salvage for RNA and DNA synthesis (3, 5-8). Erythrocytes also lack enzymes for de novo purine synthesis. The parasite requires a continuous supply of purines for RNA and DNA synthesis during its replication in the erythrocyte..Adenosine is a major purine nucleoside of human blood, but the adenosine kinase activity of P. falciparum is very low (9). Adenosine deaminase is very active in the erythrocyte and the parasite; hence deamination and subsequent phosphorolysis of the product inosine by PNP 1 yield hypoxanthine, a major purine precursor for purine salvage (Fig. 1). There is no adenosine phosphorylase activity in humans, and adenine is present at very low concentrations (10). Hypoxanthine is converted to IMP by hypoxanthine phosphoribosyltransferase activity, present in large amounts in P. falciparum (11). This path makes hypoxanthine the common precursor for all purine nucleotides in the parasite (12). Depleting Plasmodium of hypoxanthine by the addition of xanthine oxidase to culture medium prevents parasite growth (13,14). This mechanism has also evolved in vivo in the cape buffalo, where resistance to Trypanosoma brucei infections has resulted from an active serum xanthine oxidase (15).In mammals, hypoxanthine is formed from nucleosides only through phosphorolysis of inosine, the reaction catalyzed by PNP. Here we test the hypothesis that inhibition of purine nucleoside phosphorylase in infected erythrocytes will inhibit P. falciparum growth by preventing hypoxanthine production. This goal is possible through the use of transition state analogue inhibitors we have developed against both human and P. falciparum PNPs (16,17). The immucillins are purine nucleoside analogue inhibitors containing a 1-(9-deazapurin-9-yl)-1,4-dideoxy-4-iminoribitol moiety (Fig. 2). Inhibition of both human and P. falciparum PNP by immucillins prevents the utilization of inosine and deoxyinosine as hypoxanthine sources. P. falciparum parasites cultured in human erythrocytes are killed by immucillins but can be rescued by hypoxanthine and not ...

Immucillins are logically designed transition-state analogue inhibitors of mammalian purine nucleoside phosphorylase (PNP) that induce purine-less death of Plasmodium falciparum in cultured erythrocytes (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226 -3231). PNP is present at high levels in human erythrocytes and in P. falciparum, but the Plasmodium enzyme has not been characterized. A search of the P. falciparum genome data base yielded an open reading frame similar to the PNP from Escherichia coli. PNP from P. falciparum (P. falciparum PNP) was cloned, overexpressed in E. coli, purified, and characterized. The primary amino acid sequence has 26% identity with E. coli PNP, has 20% identity with human PNP, and is phylogenetically unique among known PNPs with equal genetic distance between PNPs and uridine phosphorylases. Recombinant P. falciparum PNP is catalytically active for inosine and guanosine but is less active for uridine. The immucillins are powerful inhibitors of P. falciparum PNP. Immucillin-H is a slow onset tight binding inhibitor with a K i * value of 0.6 nM. Eight related immucillins are also powerful inhibitors with dissociation constants from 0.9 to 20 nM. The K m /K i * value for immucillin-H is 9000, making this inhibitor the most powerful yet reported for P. falciparum PNP. The PNP from P. falciparum differs from the human enzyme by a lower K m for inosine, decreased preference for deoxyguanosine, and reduced affinity for the immucillins, with the exception of 5-deoxy-immucillin-H. These properties of P. falciparum PNP are consistent with a metabolic role in purine salvage and provide an explanation for the antibiotic effect of the immucillins on P. falciparum cultured in human erythrocytes.