Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases

Aminoacyl-tRNA synthetases are well known for their remarkable precision in substrate selection during aminoacyl-tRNA formation. Some synthetases enhance the accuracy of this process by editing mechanisms that lead to hydrolysis of incorrectly activated and/or charged amino acids. Prolyl-tRNA synthetases (ProRSs) can be divided into two structurally divergent groups, archaeal-type and bacterial-type enzymes. A striking difference between these groups is the presence of an insertion domain (approximately 180 amino acids) in the bacterial-type ProRS. Because the archaeal-type ProRS enzymes have been shown to recognize cysteine, we tested selected ProRSs from all three domains of life to determine whether cysteine activation is a general property of ProRS. Here we show that cysteine is activated by recombinant ProRS enzymes from the archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus, from the eukaryote Saccharomyces cerevisiae, and from the bacteria Aquifex aeolicus, Borrelia burgdorferi, Clostridium sticklandii, Cytophaga hutchinsonii, Deinococcus radiodurans, Escherichia coli, Magnetospirillum magnetotacticum, Novosphingobium aromaticivorans, Rhodopseudomonas palustris, and Thermus thermophilus. This non-cognate amino acid was efficiently acylated in vitro onto tRNA(Pro), and the misacylated Cys-tRNA(Pro) was not edited by ProRS. Therefore, ProRS exhibits a natural level of mischarging that is to date unequalled among the aminoacyl-tRNA synthetases.     

Structures of two bacterial prolyl-tRNA synthetases with and without a cis-editing domain

Prolyl-tRNA synthetases (ProRSs) are unique among synthetases in that they have diverse architectures, notably the variable presence of a cis-editing domain homologous to the freestanding deacylase proteins YbaK and ProX. Here, we describe crystal structures of two bacterial ProRSs from the pathogen Enterococcus faecalis, which possesses an editing domain, and from Rhodopseudomonas palustris, which does not. We compare the overall structure and binding mode of ATP and prolyl-adenylate with those of the archael/eukaryote-type ProRS from Thermus thermophilus. Although structurally more homologous to YbaK, which preferentially hydrolyzes Cys-tRNA(Pro), the editing domain of E. faecalis ProRS possesses key elements similar to ProX, with which it shares the activity of hydrolyzing Ala-tRNA(Pro). The structures give insight into the complex evolution of ProRSs, the mechanism of editing, and structural differences between prokaryotic- and eukaryotic-type ProRSs that can be exploited for antibiotic design.     

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA^Pro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNA^Pro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.     

Role of zinc in catalytic activity of carbonic anhydrase IX

The carbonic anhydrases (CAs) in the α class are zinc-dependent metalloenzymes. Previous studies have reported that recombinant forms of carbonic anhydrase IX (CAIX), a membrane-bound form of CA expressed in solid tumors, appear to be activated by low levels of zinc independent of its well-studied role at the catalytic site. In this study, we sought to determine if CAIX is stimulated by zinc in its native environment. MDA-MB-231 breast cancer cells express CAIX in response to hypoxia. We compared CAIX activity associated with membrane ghosts isolated from hypoxic cells with that in intact hypoxic cells. We measured CA activity directly using (18)O exchange from (13)CO(2) into water determined by membrane inlet mass spectrometry. In membrane ghosts, there was little effect of zinc at low concentrations on CAIX activity, although at high concentration zinc was inhibitory. In intact cells, zinc had no significant effect on CAIX activity. This suggests that there is an appreciable decrease in sensitivity to zinc when CAIX is in its natural membrane milieu compared to the purified forms.     

Role of the alpha-helix 163-170 in factor Xa catalytic activity

Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa alpha-helix 163-170 (h163-170), Arg(165) and Lys(169), participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH(2)-terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na(+) because using a higher Na(+) concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH(2)-terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na(+)-binding site of the protease and that residues Val(163) and Ser(167) play a key role in this interaction.     

Role of conserved amino acids in the catalytic activity of Escherichia coli primase

The role of conserved amino acid residues in the polymerase domain of Escherichia coli primase has been studied by mutagenesis. We demonstrate that each of the conserved amino acids Arg146, Arg221, Tyr230, Gly266, and Asp311 is involved in the process of catalysis. Residues Glu265 and Asp309 are also critical because a substitution of each amino acid irreversibly destroys the catalytic activity. Two K229A and M268A mutant primase proteins synthesize only 2-nucleotide products in de novo synthesis reactions under standard conditions. Y267A mutant primase protein synthesizes both full-size and 2-nucleotide RNA, but with no intermediate-size products. From these data we discuss the significant step of the 2-nucleotide primer RNA synthesis by E. coli primase and the role of amino acids Lys229, Tyr267, and Met268 in primase complex stability.     

ATPase activity of non-ribosomal peptide synthetases

Adenylation domains of non-ribosomal peptide synthetases (NRPS) catalyse the formation of aminoacyl adenylates, and in addition synthesize mono- and dinucleoside polyphosphates. Here, we show that NRPS systems furthermore contain an ATPase activity in the range of up to 2 P(i)/min. The hydrolysis rate by apo-tyrocidine synthetase 1 (apo-TY1) is enhanced in the presence of non-cognate amino acid substrates, correlating well with their structural features and the diminishing adenylation efficiency. A comparative analysis of the functional relevance of an analogous sequence motif in P-type ATPases and adenylate kinases (AK) allowed a putative assignment of the invariant aspartate residue from the TGDLA(V)R(K) core sequence in NRPS as the Mg(2+) binding site. Less pronounced variations in ATPase activity are observed in domains with relaxed amino acid specificity of gramicidin S synthetase 2 (GS2) and delta-(L-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), known to produce a set of substitutional variants of the respective peptide product. These results disclose new perspectives about the mode of substrate selection by NRPS.     

Processivity of translation in the eukaryote cell: Role of aminoacyl-tRNA synthetases

Several lines of evidence led to the conclusion that mammalian ribosomal protein synthesis is a highly organized biological process in vivo. A wealth of data support the concept according to which tRNA aminoacylation, formation of the ternary complex on EF1A and delivery of aminoacyl-tRNA to the ribosome is a processive mechanism where tRNA is vectorially transferred from one component to another. Polypeptide extensions, referred to as tRBDs (tRNA binding domains), are appended to mammalian and yeast aminoacyl-tRNA synthetases. The involvement of these domains in the capture of deacylated tRNA and in the sequestration of aminoacylated tRNA, suggests that cycling of tRNA in translation is mediated by the processivity of the consecutive steps. The possible origin of the tRBDs is discussed.     

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin''s in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.     

The nature of the catalytic domain of 2'-5'-oligoadenylate synthetases.

2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol beta, P69 can assume an alphabetabetaalphabetabetabeta structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes. Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.     

Role of surface residue 184 in the catalytic activity of NADH oxidase from Streptococcus pyogenes

Nicotinamide adenine dinucleotide (NADH) oxidase from Streptococcus pyogenes (SpNox) is a flavoprotein harboring one molecule of noncovalently bound flavin adenine dinucleotide. It catalyzes the oxidation of NADH by reducing molecular O₂ to H₂O directly through a four-electron reduction. In this study, we selected the lysine residues on the surface of SpNox and mutated them into arginine residues to study the effect on the enzyme activity. A single-point mutation (K184R) at the surface of SpNox enhanced NADH oxidase activity by approximately 50 % and improved thermostability with 46.6 % longer half life at 30 °C. Further insights into the function of residue K184 were obtained by substituting it with other nonpolar, polar, positively charged, and negatively charged residues. To elucidate the role of this residue, computer-assisted molecular modeling and substrate docking were performed. The results demonstrate that even a single mutation at the surface of the enzyme induces changes in the interaction at the active site and affects the activity and stability. Additionally, the data also suggest that the K184R mutant can be used as an effective biocatalyst for NAD⁺ regeneration in l-rare sugar production.     

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through beta-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords approximately 10-fold higher K(m) values but no significant change in V(max), consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage.     

Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.     

Inhibition of the activity of aminoacyl-tRNA synthetases in pollen grains of maize

Extracts from pollen grains of maize ( Zea mays L.) show a low activity of aminoacyltRNA synthetases (EC 6. 1. 1). They also contain a specific factor inhibiting the activity of these enzymes. The molecular mass of this factor, which may be a short peptide, is about 3000 Da as determined by column chromatography on Sephadex G-25 Fine. The Michaelis constant (K_m), determined for the amino acid in the presence of this factor, suggests its allosteric influence on the affinity of the enzyme. Short-term incubation of the factor with pronase R resulted in conversion of the inhibiting action into a stimulating one. Kinetics of aminoacylation reactions confirm inhibitory and stimulative influences of the effector on the enzyme activity. High performance liquid chromatography shows that inhibition of the activity of aminoacyl-tRNA synthetases is affected by a group of compounds of similar molecular masses.     

Role of zinc ion for catalytic activity in d-serine dehydratase from Saccharomyces cerevisiae

d-Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme catalyzing d-serine dehydration. The enzyme contains 1 mol Zn(2+) in its active site and shows a unique zinc dependence. The Zn(2+) is essential for the d-serine dehydration, but not for the α,β-elimination of β-Cl-d-alanine catalyzed as a side-reaction. The fact that dehydration of d-threonine and d-allo-threonine, also catalyzed by DsdSC, is likewise Zn(2+) dependent indicates that Zn(2+) is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of C(β) . Removal of Zn(2+) results in a less polar active site without changing the gross conformation of DsdSC. (1) H NMR determined the rates of α-hydrogen abstraction and hydroxyl group elimination of d-serine in (2) H(2) O to be 9.7 and 8.5 s(-1) , respectively, while the removal of Zn(2+) abolished both reactions. Mutation of Cys400 or His398 within the Zn(2+) binding sites to Ala endowed DsdSC with similar properties to those of the Zn(2+) -depleted wild-type enzyme: the mutants lost the reactivity toward d-serine and d-threonine but retained that toward β-Cl-d-alanine. (1) H NMR analysis also revealed that both α-hydrogen abstraction and hydroxyl group elimination from d-serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the α-hydrogen abstraction and hydroxyl group elimination in a concerted fashion.     

Aminoacyl-tRNA synthetases activity as a growth index in zooplankton

A simple method for the assay of aminoacyl-tRNA synthetases(AARS) activity was modified for application in planktonic crustaceansas an index of somatic growth. The cladoceran Daphnia magnawas cultured in the laboratory and its AARS activity measuredwithout substrate addition. The relationship between the enzymeactivities of animals of similar age and individual biomassgrowing at different rates was tested. A significant relationshipwas found between AARS activity and somatic growth in termsof both protein and dry weight.     

Species-specific differences in the regulation of the aminoacylation activity of mammalian tryptophanyl-tRNA synthetases

Tryptophanyl-tRNA synthetases (TrpRSs) catalyze the aminoacylation of tRNA^Trp. Previously, I demonstrated that Zn²⁺-depleted human TrpRS is enzymatically inactive and that binding of Zn²⁺ or heme to human TrpRS stimulates its aminoacylation activity. In the present study, bovine and mouse TrpRSs were found to be constitutively active regardless of the presence of Zn²⁺ or ferriprotoporphyrin IX chloride. Mutagenesis experiments demonstrated that the human H130R mutant is constitutively active and that the bovine R135H, E438A double mutant binds with Zn²⁺ or heme to enhance its aminoacylation activity as does human wild-type TrpRS. These results provide the first evidence of species-specific regulation of TrpRS activity.     

Comparison of the catalytic roles played by the KMSKS motif in the human and Bacillus stearothermophilus trosyl-tRNA synthetases

The Class I aminoacyl-tRNA synthetases are characterized by two signature sequence motifs, "HIGH" and "KMSKS." In Bacillus stearothermophilus tyrosyl-tRNA synthetase, the KMSKS motif (230KFGKT234) has been shown to stabilize the transition state for tyrosine activation through interactions with the pyrophosphate moiety of ATP. In most eukaryotic tyrosyl-tRNA synthetases, the second lysine in the KMSKS motif is replaced by a serine or an alanine residue. Recent kinetic studies indicate that potassium functionally compensates for the absence of the second lysine in the human tyrosyl-tRNA synthetase (222KKSSS226). In this paper, site-directed mutagenesis and pre-steady state kinetics are used to determine the roles that serines 224, 225, and 226 play in catalysis of the tyrosine activation reaction. In addition, the catalytic role played by a downstream lysine conserved in eukaryotic tyrosyl-tRNA synthetases, Lys-231, is investigated. Replacing Ser-224 and Ser-226 with alanine decreases the forward rate constant 7.5- and 60-fold, respectively. In contrast, replacing either Ser-225 or Lys-231 with alanine has no effect on the catalytic activity of the enzyme. These results are consistent with the hypothesis that the KMSSS sequence in human tyrosyl-tRNA synthetase stabilizes the transition state for the tyrosine activation reaction by interacting with the pyrophosphate moiety of ATP. In addition, although they play similar roles in catalysis, the overall contribution of the KMSKS motif to catalysis appears to be significantly less in human tyrosyl-tRNA synthetase than it is in the B. stearothermophilus enzyme.