Coevolution of RtcB and Archease created a multiple-turnover RNA ligase.

RtcB is a noncanonical RNA ligase that joins either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. The genes encoding RtcB and Archease constitute a tRNA splicing operon in many organisms. Archease is a cofactor of RtcB that accelerates RNA ligation and alters the NTP specificity of the ligase from Pyrococcus horikoshii. Yet, not all organisms that encode RtcB also encode Archease. Here we sought to understand the differences between Archease-dependent and Archease-independent RtcBs so as to illuminate the evolution of Archease and its function. We report on the Archease-dependent RtcB from Thermus thermophilus and the Archease-independent RtcB from Thermobifida fusca. We find that RtcB from T. thermophilus can catalyze multiple turnovers only in the presence of Archease. Remarkably, Archease from P. horikoshii can activate T. thermophilus RtcB, despite low sequence identity between the Archeases from these two organisms. In contrast, RtcB from T. fusca is a single-turnover enzyme that is unable to be converted into a multiple-turnover ligase by Archease from either P. horikoshii or T. thermophilus. Thus, our data indicate that Archease likely evolved to support multiple-turnover activity of RtcB and that coevolution of the two proteins is necessary for a functional interaction.

Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme.

A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation.

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3'-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.

Structures of the noncanonical RNA ligase RtcB reveal the mechanism of histidine guanylylation.

RtcB is an atypical RNA ligase that joins either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. In contrast to typical RNA ligases, which rely on ATP and Mg(II), catalysis by RtcB is dependent on GTP and Mn(II) with ligation proceeding through a covalent RtcB-histidine-GMP intermediate. Here, we present three structures of Pyrococcus horikoshii RtcB complexes that capture snapshots along the entire guanylylation pathway. These structures show that prior to binding GTP, a single manganese ion (Mn1) is bound to RtcB. To capture the step immediately preceding RtcB guanylylation, we determined a structure of RtcB in complex with Mn(II) and the unreactive GTP analogue guanosine 5'-(α-thio)triphosphate (GTPαS). This structure shows that Mn1 is poised to stabilize the pentavalent transition state of guanylylation while a second manganese ion (Mn2) is coordinated to a nonbridging oxygen of the γ-phosphoryl group. The pyrophosphate leaving group of GTPαS is oriented apically to His404 with the ε-nitrogen poised for in-line attack on the α-phosphorus atom. The structure of RtcB in complex with GTPαS also reveals the network of hydrogen bonds that recognize GTP and illuminates the significant conformational changes that accompany the binding of this cofactor. Finally, a structure of the enzymic histidine-GMP intermediate depicts the end of the guanylylation pathway. The ensuing molecular description of the RtcB guanylylation pathway shows that RtcB and classical ATP- and Mg(II)-dependent nucleic acid ligases have converged upon a similar two-metal mechanism for formation of the nucleotidylated enzyme intermediate.

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate.

Microbial niches contain toxic chemicals capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival in the presence of a normally lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. We also screened a comprehensive library of E. coli single-gene deletion mutants and identified 63 strains displaying increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene encoding a predicted glutathione transferase. Herein, YliJ is shown to catalyze the glutathione-dependent dehalogenation of bromoacetate with a k(cat)/K(m) value of 5.4 × 10(3) M(-1) s(-1). YliJ displays exceptional substrate specificity and produces a rate enhancement exceeding 5 orders of magnitude, remarkable characteristics for reactivity with a nonnatural molecule. This study illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins.

tRNA ligase catalyzes the GTP-dependent ligation of RNA with 3'-phosphate and 5'-hydroxyl termini.

The RNA ligase RtcB is conserved in all domains of life and is essential for tRNA maturation in archaea and metazoa. Here we show that bacterial and archaeal RtcB catalyze the GTP-dependent ligation of RNA with 3'-phosphate and 5'-hydroxyl termini. Reactions with analogues of RNA and GTP suggest a mechanism in which RtcB heals the 3'-phosphate terminus by forming a 2',3'-cyclic phosphate before joining it to the 5'-hydroxyl group of a second RNA strand. Thus, RtcB can ligate RNA cleaved by RNA endonucleases, which generate 2',3'-cyclic phosphate and then 3'-phosphate termini on one strand, and a 5'-hydroxyl terminus on another strand.

L-glyceraldehyde 3-phosphate reductase from Escherichia coli is a heme binding protein.

Recently, we reported that YghZ from Escherichia coli functions as an efficient L-glyceraldehyde 3-phosphate reductase (Gpr). Here we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a K(d) value of 5.8+/-0.2 microM in the absence of NADPH and a K(d) value of 11+/-1.3 microM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the L-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K+ channel beta-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K+ channels with hemoprotein-like, O2-sensing properties. To date the molecular origin of O2 sensing by voltage-gated K+ channels is unknown and the results presented herein suggest a role for heme in this process.

A metabolic bypass of the triosephosphate isomerase reaction.

Triosephosphate isomerase (TIM) catalyzes the interconversion of d-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the Escherichia coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of l-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate.

Coordinated peak expression of MMP-26 and TIMP-4 in preinvasive human prostate tumor.

The identification of novel biomarkers for early prostate cancer diagnosis is highly important because early detection and treatment are critical for the medical management of patients. Disruption in the continuity of both the basal cell layer and basement membrane is essential for the progression of high-grade prostatic intraepithelial neoplasia (HGPIN) to invasive adenocarcinoma in human prostate. The molecules involved in the conversion to an invasive phenotype are the subject of intense scrutiny. We have previously reported that matrix metalloproteinase-26 (MMP-26) promotes the invasion of human prostate cancer cells via the cleavage of basement membrane proteins and by activating the zymogen form of MMP-9. Furthermore, we have found that tissue inhibitor of metalloproteinases-4 (TIMP-4) is the most potent endogenous inhibitor of MMP-26. Here we demonstrate higher (p<0.0001) MMP-26 and TIMP-4 expression in HGPIN and cancer, compared to non-neoplastic acini. Their expression levels are highest in HGPIN, but decline in invasive cancer (p<0.001 for each) in the same tissues. Immunohistochemical staining of serial prostate cancer tissue sections suggests colocalization of MMP-26 and TIMP-4. The present study indicates that MMP-26 and TIMP-4 may play an integral role during the conversion of HGPIN to invasive cancer and may also serve as markers for early prostate cancer diagnosis.