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Bedford C.A., Challis J.R.G., Harrison F.A., Heap. R.B.: The role of oestrogens and progesterone in the onset of parturition in various species. J. Reprod. Fer-.
Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, NJ 07103, USA; [email protected] or [email protected]; Tel.: +1-973-972-8733 Department of Biochemistry and Biotechnology, University of Life Sciences, Poznan 60-632, Poland Presented at the Banbury Center, Cold Spring Harbor Laboratory, NY meeting on “Evolution of the Translational Apparatus and implication for the origin of the Genetic Code”, 13–16 November 2016.
Figure 1. Chemical activation and information transfer transfer by synthetases (AARSs). Figure 1. Chemical activation and information byaminoacyl-tRNA aminoacyl-tRNA synthetases (AARSs).
ago  (these timelines are based on counting relative node, i.e., branch point, distance in the rooted Life 2017, 7, 6 25 trees and using a molecular clock of protein folds to convert relative age into geological3 oftime ). Because of Phylogenetic their crucial role in the translation maintenance of the Genetic Code, analysis [11,31]. analyses of structural domains and present in proteomes  suggest that catalytic of AARSs belong to the oldest fold families and may have appearedcan about 3.7 billion years into the of AARSs domains structures and mechanisms of reactions catalyzed by AARSs provide insights while separate that edit misacylated tRNA appeared later,examines about 3.2 billion years ago origin andago, evolution of thedomains Genetic Code [35,37]. The present article the links between Hcy  (these timelines are based on counting relative node, i.e., branch point, distance in the rooted editing, thioester chemistry, and the origin of the amino acid activation for the coded protein synthesis. trees and using a molecular clock of protein folds to convert relative age into geological time ). Available dataBecause suggest thatcrucial ancestral wereand involved in the thiol (coenzyme A, pantetheine) of their role inAARSs the translation maintenance of the Genetic Code, analysis of AARSs reactions structures and mechanisms of reactions catalyzed by AARSs synthesis can providebefore insightsthe intoemergence the aminoacylation and thioester-based non-coded peptide of origin andand evolution of the Genetic Code [35,37]. The present article examines the links between Hcy the Genetic Code the ribosomal protein biosynthetic machinery. 2.
Misactivated Hcy is edited by an intramolecular reaction between the side chain thiolate and the Misactivated Hcy is edited by an thioester intramolecular reaction between(Reaction the side chain thiolate and the activated carboxyl of Hcy, affording the Hcy-thiolactone (2)) [18,38]. activated carboxyl of Hcy, affording the thioester Hcy-thiolactone (Reaction II) [18,38].
thiolate of Hcy molecule is a nucleophile, to accomplish editing. Editing reactions of all other amino Hcy editing is unique in that it involves an intramolecular Reaction (II), in which the side chain acids, including a related thio-amino acid Cys, are intermolecular and use water hydroxide as a thiolate of Hcy molecule is a nucleophile, to accomplish editing. Editing reactions of all other amino nucleophile . Hcy is edited by the cyclization to Hcy-thiolactone at the synthetic/editing catalytic acids, including a related thio-amino acid Cys, are intermolecular and use water hydroxide as a site in the Rossman-fold domain of class I AARS [55,56] and at the β catalytic domain of Class II nucleophile . Hcy is edited by the cyclization to Hcy-thiolactone at sheet the synthetic/editing catalytic LysRS [29,38]. site in the Rossman-fold domain of class I AARS [55,56] and at the β sheet catalytic domain of Class II LysRS [29,38].
A model for pre-transfer Hcy editing explains how MetRS partitions Met and Hcy between the model for pre-transfer Hcy editing explains MetRS partitions Met and crystal Hcy between the , syntheticAand editing pathways, respectively. The how model is supported by the structure synthetic and editing pathways, respectively. The model is supported by the crystal structure , structure/function [55,56], and computational  studies of E. coli MetRS. In the synthetic pathway, 0 -hydroxyl 0 -terminus structure/function and computational studies ofof E.the coli 3MetRS. In theof synthetic pathway, the activated carboxyl[55,56], of Met reacts with the 2 tRNAMet, affording the activated carboxyl of Met reacts with the 2′-hydroxyl of the 3′-terminus of tRNAMet, affording Met-tRNAMet. In the editing pathway, the activated carboxyl of Hcy reacts with the thiolate of its side Met-tRNAMet. In the editing pathway, the activated carboxyl of Hcy reacts with the thiolate of its chain, affording Hcy~ thiolactone. side chain, affording Hcy~thiolactone. Whether an amino acid substrate completes the synthetic or editing pathway is determined by Whether an amino acid substrate completes the synthetic or editing pathway is determined by the partitioning of its side chain specificityand andthiol-binding thiol-binding subsites Met completes the partitioning of its side chainbetween between the the specificity subsites .. Met completes the synthetic pathway because is bound boundby bythe thehydrophobic hydrophobic hydrogen-bonding the synthetic pathway becauseits itsside side chain chain is andand hydrogen-bonding interactions with W305 and Y15 specificitysub-site sub-site (Figure interactions with W305 and Y15residues residuesin in the the specificity (Figure 2). 2).
Figure 2. The synthetic/editingactive active site site of synthetase (MetRS): Hydrophobic Figure 2. The synthetic/editing ofE.E.coli colimethionyl-tRNA methionyl-tRNA synthetase (MetRS): Hydrophobic and hydrogen-bonding interactions provide specificity for the cognate substrate L-methionine. and hydrogen-bonding interactions provide specificity for the cognate substrate L-methionine. Superimposition of Cα carbon atoms for the MetRS·Met complex (beige) and free MetRS (light grey), Superimposition of Cα carbon atoms for the MetRS·Met complex (beige) and free MetRS (light grey), solved at 1.8 Å resolution, shows movements of active site residues upon binding of Met. Residue solved at 1.8 Å resolution, shows movements of active site residues upon binding of Met. Residue colors are red in the MetRS·Met complex and green in free MetRS, and L-methionine is magenta. colors are redwith in the MetRS·from Met complex Reprinted permission ref. . and green in free MetRS, and L-methionine is magenta. Reprinted with permission from reference .
In contrast, contrast, the side chain of Hcy, Hcy, missing missing the the methyl methyl group group of of Met, Met, interacts interacts weakly weakly with with the the In contrast, theThis side allows chain of Hcy, methyl groupwith of Met, weakly with the specificity sub-site. the sidemissing chain ofthe Hcy to interact D259interacts  in the thiol-binding  in the thiol-binding specificity sub-site. allowsediting the side of Hcyto toHcy-thiolactone interact with D259  in thiol-binding sub-site , which facilitates by cyclization (Figure 3). Consistent with , whichThis facilitates editing bychain cyclization to Hcy-thiolactone (Figure 3).the Consistent with sub-site , which facilitates editing by cyclization to Hcy-thiolactone (Figure 3). Consistent with this model, mutations of W305 and Y15 residues, which form the hydrophobic Met-binding sub-site, this model, mutationsdiscrimination of W305 and Y15 residues, which reduce the discrimination by the the enzyme . form the hydrophobic Met-binding sub-site, reduce the Hcy/Met Hcy/Met by enzyme reduce the Hcy/Met discrimination by the enzyme .
Figure 3. 3. Editing Editing of of miscativated miscativated homocysteine homocysteine (Hcy~AMP) (Hcy~AMP) at at the the catalytic catalytic module module of of an an AARS: AARS: The The Figure Figure 3. Editing of miscativated homocysteine adenylate (Hcy~AMP) at the catalytic module and of anAMP, AARS: The MetRS-catalyzed cyclization of homocysteinyl to form Hcy-thiolactone which MetRS-catalyzed cyclization of homocysteinyl adenylate to form Hcy-thiolactone and AMP, which are MetRS-catalyzed cyclization of homocysteinyl adenylate to form Hcy-thiolactone and AMP, which are subsequently released the synthetic/editing active of MetRS. subsequently released fromfrom the synthetic/editing active site site of MetRS. are subsequently released from the synthetic/editing active site of MetRS.
Computational studies  suggest that D259 plays an essential role as a mechanistic base that studies  suggest that D259 plays an essential role as a mechanistic base that Computational studies deprotonates the side chain thiol in Hcy~AMP at the catalytic module of MetRS (Figure 4). In the deprotonates the side chain thiol in in Hcy~AMP at thethe catalytic module of MetRS (Figure 4). In 4). the In initial deprotonates the side chain thiol Hcy~AMP module of MetRS (Figure the initial MetRS·Hcy~AMP complex the distance at SHcy···Ocatalytic Asp259 (5.30 Å ) is markedly shorter than the MetRS·MetRS· Hcy~AMP complex the distance SHcy ···OSAsp259 (5.30 is markedly shorter than the than distance initial Hcy~AMP complex the distance Hcy···O Asp259 Å) (5.30 Å ) is markedly shorter the distance SHcy···Ophos in Hcy~AMP (7.15 Å ) (Table 1). The rate-limiting step in Hcy-thiolactone SHcy ···Ophos Hcy~AMP (7.15 Å) (7.15 (TableÅ 1). The rate-limiting step−1 in Hcy-thiolactone formation distance S Hcyin · · · O phos in Hcy~AMP ) (Table 1). The rate-limiting step in Hcy-thiolactone formation is the rotation about the Cβ−Cγ bond with 27.5 kJ·mol energy barrier. This is more −1 energy is the rotation about the Cabout −Cγthe bond with 27.5 kJ ·mol27.5 is more formation is the rotation C β−C γ bond with kJ· mol−1barrier. energyThis barrier. Thisfavorable is which more β −1 favorable than 98.2−51 kJ·mol energy barrier for an alternative substrate-assisted mechanism in −1 than 98.25than kJ·mol energy barrier for an for alternative substrate-assisted mechanism in which the favorable 98.2 5 kJ· mol energy barrier an alternative substrate-assisted mechanism in which the non-bridging oxygen of phosphate in Hcy~AMP acts as a base. non-bridging oxygen of phosphate in Hcy~AMP actsacts as aas base. the non-bridging oxygen of phosphate in Hcy~AMP a base.
Figure 4. Energetics of Hcy~AMP editing at the catalytic module of MetRS. Asp259 is a mechanistic Figure 4. Energetics of Hcy~AMP editing at catalytic Reproduced module of MetRS. Asp259 is afrom mechanistic base that deprotonates the side chain thiol inthe Hcy~AMP. with permission ref. base . Figure 4. Energetics of Hcy~AMP editing at the catalytic module of MetRS. Asp259 is a mechanistic base that deprotonates the side chain thiol in Hcy~AMP. Reproduced with permission from ref. . that deprotonates the side chain thiol in Hcy~AMP. Reproduced with permission from reference .
activity of MetRS. MetRS. When When the active site is occupied Figure 5. Amino acid:coenzyme A (CoA-SH) ligase activity by Met-tRNA Met-tRNAoror Met-AMP, the thiol subsite is occupied by CoA-SH, MetRS the catalyzes the by Met-AMP, andand the thiol subsite is occupied by CoA-SH, MetRS catalyzes formation formation of Met-S-CoA thioester. An R represents the bulk of CoA-SH or other thiol molecule. of Met-S-CoA thioester. An R represents the bulk of CoA-SH or other thiol molecule.
2.2.2. Leucyl-tRNA Leucyl-tRNA Synthetase Synthetase (LeuRS), (LeuRS), Isoleucyl-tRNA Isoleucyl-tRNA Synthetase Synthetase (IleRS), (IleRS), Valyl-tRNA Valyl-tRNA Synthetase 2.2.2. (ValRS) Synthetase (ValRS) A similar model explains Hcy editing [25,26] by related Class I AARS. ValRS, LeuRS, and IleRS have active activesites, sites,D490, D490, E532, E550, respectively, that correspond ofand MetRS and are have E532, andand E550, respectively, that correspond to D359toofD359 MetRS are similarly similarly positioned with tocenter the Ccarb center of the substrate Hcy~AMP . Computational positioned with respect to respect the Ccarb of the substrate Hcy~AMP . Computational analyses analyses that the Hcy~AMP in theirsites active in aconformation linear conformation show thatshow the Hcy~AMP substratesubstrate binds in binds their active in asites linear similar similar to that to that observed for MetRS. The average C carb · · · O Asp/Glu distances are within 1.16 Å of each other observed for MetRS. The average Ccarb ···OAsp/Glu distances are within 1.16 Å of each other whereas all all distances SHcy···Ccarbare distances are within 1.24 Å of(Table each 1). other (Table 1). Moreforimportantly, for Swhereas within 1.24 Å of each other More importantly, MetRS, LeuRS, Hcy ···Ccarb MetRS, LeuRS, and ValRS, Hcy···OAsp/Glu distance is significantly shorter thanSthe and ValRS, the average SHcythe ···Oaverage distance is significantly shorter than the average ···Ophos Hcy average Asp/Glu S S Hcy · · · O phos distance by 2.15 Å , 2.27 Å , and 2.06 Å , respectively. Thus, each of these Asp/Glu residues distance by 2.15 Å, 2.27 Å, and 2.06 Å, respectively. Thus, each of these Asp/Glu residues can act as a can act as abase mechanistic base that the deprotonates the side chain thiol in facilitate Hcy~AMP and facilitate mechanistic that deprotonates side chain thiol in Hcy~AMP and Hcy-thiolactone Hcy-thiolactone formation MetRS, and itValRS. it is Glu550 less clear formation by MetRS, LeuRS,byand ValRS.LeuRS, However, is less However, clear whether canwhether act as a Glu550 base in can act as a base formation in Hcy-thiolactone Hcy-thiolactone catalyzedformation by IleRS. catalyzed by IleRS. 2.2.3. Lysyl-tRNA Lysyl-tRNA Synthetase Synthetase (LysRS) (LysRS)Edits EditsHomocysteine Homocysteine(Hcy), (Hcy),Ornithine Ornithine(Orn), (Orn),Homoserine Homoserine(Hse), but Mischarges tRNALys withLysProteinogenic AminoAmino Acids Acids (Hse), but Mischarges tRNA with Proteinogenic Hcy editing occurs also at the β β sheet catalytic domain of Class II E. E. coli coli LysRS , which which also Lys Lys Lys Lys edits Orn and Hse. Editing by LysRS is not affected by tRNA and there is no tRNA mischarging Orn and Hse. Editing by LysRS is not affected by tRNA and there is no tRNA Lys with several other amino acids (Arg, Thr, with with Hcy Hcy or or Orn . However, LysRS LysRS mischarges mischarges tRNA tRNALys with several other amino acids (Arg, Thr, Lys , Thr-tRNA Lys , and Lys, Thr-tRNA Lys, Met, Cys, Ser) andand doesdoes not deacylate mischarged Arg-tRNA Cys, Leu, Leu,Ala, Ala,oror Ser) not deacylate mischarged Arg-tRNA Lys Lys Lys Lys Met-tRNA . Recent data show that Met-tRNA (and other tRNAs mischarged with Met) are and Met-tRNA . Recent data show that Met-tRNA (and other tRNAs mischarged with Met) formed in E. E. coli coli and and mammalian mammalian cells cells in in response response to to stress stress conditions conditions[13,14]. [13,14].
3. Expanding Expanding the the Genetic Genetic Code: Code: Decoding Decoding Methionine Methionine Codons Codons by by Homocysteine Homocysteine 3. By preventing preventing the Hcy to tRNA, the the editing editing reaction reaction assures assures that excluded By the attachment attachment of of Hcy to tRNA, that Hcy Hcy is is excluded from the Genetic Code. Weak interactions of the side chain of Hcy with the specificity subsite allow from the Genetic Code. Weak interactions of the side chain of Hcy with the specificity subsite allow binding of of the the Hcy Hcy side side chain chain to to the the thiol-binding thiol-binding subsite subsite in in the the catalytic catalytic domain domain of of MetRS MetRS [55,56]. [55,56]. binding Thus, modifications of the side chain of Hcy that increase binding to the specificity subsite should Thus, modifications of the side chain of Hcy that increase binding to the specificity subsite should Met This is achieved by S-nitrosylation of Hcy to prevent prevent editing editing and and facilitate facilitate the the transfer transfer to to tRNA tRNAMet.. This is achieved by S-nitrosylation of Hcy to S-nitroso-Hcy (S-NO-Hcy), which binds to the MetRS with affinity10-fold 10-foldgreater greater than Hcy and S-nitroso-Hcy (S-NO-Hcy), which binds to the MetRS with affinity than Hcy and is is activated by MetRS to form S-NO-Hcy~AMP . In contrast to Hcy-AMP which is edited, activated by MetRS to form S-NO-Hcy~AMP . In contrast to Hcy-AMP which is edited, S-NO-Hcy~AMP subsite which leads to S-NO-Hcy~AMP is is resistant resistantto toediting editingdue duetotostronger strongerbinding bindingtotothe thespecificity specificity subsite which leads Met Met thethe transfer of S-NO-Hcy to tRNA , affording S-NO-Hcy~tRNA to transfer of S-NO-Hcy to tRNAMet , affording S-NO-Hcy~tRNAMet(Figure (Figure6). 6).
Figure by MetRS MetRS . . Figure 6. 6. Aminoacylation Aminoacylation of of tRNA tRNA with with S-NO-Hcy S-NO-Hcy catalyzed catalyzed by Met has a similar susceptibility to spontaneous deacylation as Met~tRNAMet The S-NO-Hcy-tRNAMet Met The S-NO-Hcy-tRNA has a similar susceptibility to spontaneous deacylation as Met~tRNA Met affords Met, the least with a half-life of 28 min. De-nitrosylation of S-nitroso-Hcy~tRNAMet Hcy~tRNAMet with a half-life of 28 min. De-nitrosylation of S-nitroso-Hcy~tRNA affords Hcy~tRNA , the least stable aminoacyl-tRNA known, which spontaneously deacylates with a half-life of 15 s to form stable aminoacyl-tRNA known, which spontaneously deacylates with a half-life of 15 s to form Met. Hcy-thiolactone and free tRNAMet Hcy-thiolactone and free tRNA . Met As expected, S-NO-Hcy~tRNAMet is a substrate for protein synthesis on ribosomes, which allows As expected, S-NO-Hcy~tRNA is a substrate for protein synthesis on ribosomes, which allows translational incorporation of S-NO-Hcy into protein at positions normally occupied by Met . translational incorporation of S-NO-Hcy into protein at positions normally occupied by Met . For example, when cultures of E. coli metE mutant cells (unable to metabolize Hcy to Met) expressing For example, when cultures of E. coli metE mutant cells (unable to metabolize Hcy to Met) expressing mouse dihydrofolate reductase (DHFR) protein were supplemented with S-NO-Hcy, the DHFR mouse dihydrofolate reductase (DHFR) protein were supplemented with S-NO-Hcy, the DHFR protein protein was found to contain Hcy. Control experiments in which E. coli metE cultures were was found to contain Hcy. Control experiments in which E. coli metE cultures were supplemented with supplemented with Hcy or Hcy-thiolactone, instead of S-NO-Hcy, show that there is no incorporation Hcy or Hcy-thiolactone, instead of S-NO-Hcy, show that there is no incorporation of Hcy into bacterial of Hcy into bacterial proteins . Globin and luciferase, produced in an in vitro mRNA-programmed proteins . Globin and luciferase, produced in an in vitro mRNA-programmed rabbit reticulocyte rabbit reticulocyte protein synthesis system supplemented with S-NO-Hcy~tRNAMet contain Hcy at Met protein synthesis system supplemented with S-NO-Hcy~tRNA contain Hcy at positions normally positions normally occupied by Met. occupied by Met. Translationally incorporated Hcy has also been identified in protein from cultured human Translationally incorporated Hcy has also been identified in protein from cultured human vascular vascular endothelial cells (HUVECs) (Table 2), which endogenously produce nitric oxide and Sendothelial cells (HUVECs) (Table 2), which endogenously produce nitric oxide and S-nitroso-Hcy . nitroso-Hcy . Translationally incorporated Hcy is resistant to Edman degradation whereas postTranslationally incorporated Hcy is resistant to Edman degradation whereas post-translationally translationally incorporated Hcy (by the reaction of Hcy-thiolactone with protein lysine residues ) incorporated Hcy (by the reaction of Hcy-thiolactone with protein lysine residues ) is not (Table 2, is not (Table 2, last row) last, which allows to distinguish between the two mechanisms . last row) last, which allows to distinguish between the two mechanisms . Taken together, these findings show that Hcy can gain an access to the Genetic Code by nitric oxide-mediated invasion of the methionine-coding pathway .
Table 2. Translational and post-translational incorporation of Hcy into HUVEC protein [60,62].
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