吡咯赖氨酸：第 22 种参与蛋白质生物合成的氨基酸

吡咯赖氨酸在产甲烷菌的甲胺甲基转移酶中发现,是目前已知的第 22 种参与蛋白质生物合成的氨基酸,与标准氨基酸不同的是,它由终止密码子 UAG 的有义编码形成 . 与之对应的在产甲烷菌中也含有特异的吡咯赖氨酰 -tRNA 合成酶 (PylRS) 和吡咯赖氨酸 tRNA (tRNA^Pyl). tRNA^Pyl具有不同于经典 tRNA 的特殊结构 . 产甲烷菌通过直接途径和间接途径这两种途径生成吡咯赖氨酰 -tRNA^Pyl(Pyl-tRNA^Pyl) ,它还可能通过 mRNA 上的特殊结构以及其他还未发现的机制,控制 UAG 编码成为终止密码子或者吡咯赖氨酸 . 比较了吡咯赖氨酸与另一种非标准氨基酸,第 21 种氨基酸———硒代半胱氨酸的相似点与不同点 .     

PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

Pyrrolysine is represented by an amber codon in genes encoding proteins such as the methylamine methyltransferases present in some Archaea and Bacteria. Pyrrolysyl-tRNA synthetase (PylRS) attaches pyrrolysine to the amber-suppressing tRNA^Pyl. Archaeal PylRS, encoded by pylS, has a catalytic C-terminal domain but an N-terminal region of unknown function and structure. In Bacteria, homologs of the N- and C-terminal regions of archaeal PylRS are respectively encoded by pylSn and pylSc. We show here that wild type PylS from Methanosarcina barkeri and PylSn from Desulfitobacterium hafniense bind tRNA^Pyl in EMSA with apparent K_d values of 0.12 and 0.13 μm, respectively. Truncation of the N-terminal region of PylS eliminated detectable tRNA^Pyl binding as measured by EMSA, but not catalytic activity. A chimeric protein with PylSn fused to the N terminus of truncated PylS regained EMSA-detectable tRNA^Pyl binding. PylSn did not bind other D. hafniense tRNAs, nor did the competition by the Escherichia coli tRNA pool interfere with tRNA^Pyl binding. Further indicating the specificity of PylSn interaction with tRNA^Pyl, substitutions of conserved residues in tRNA^Pyl in the variable loop, D stem, and T stem and loop had significant impact in binding, whereas those having base changes in the acceptor stem or anticodon stem and loop still retained the ability to complex with PylSn. PylSn and the N terminus of PylS comprise the protein superfamily TIGR03129. The members of this family are not similar to any known RNA-binding protein, but our results suggest their common function involves specific binding of tRNA^Pyl.     

Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase

Pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase (aaRS) recently found in some methanogenic archaea and bacteria, recognizes an unusually large lysine derivative, l-pyrrolysine, as the substrate, and attaches it to the cognate tRNA (tRNA^Pyl). The PylRS-tRNA^Pyl pair interacts with none of the endogenous aaRS-tRNA pairs in Escherichia coli, and thus can be used as a novel aaRS-tRNA pair for genetic code expansion. The crystal structures of the Methanosarcina mazei PylRS revealed that it has a unique, large pocket for amino acid binding, and the wild type M. mazei PylRS recognizes the natural lysine derivative as well as many lysine analogs, including N^?-(tert-butoxycarbonyl)-l-lysine (Boc-lysine), with diverse side chain sizes and structures. Moreover, the PylRS only loosely recognizes the α-amino group of the substrate, whereas most aaRSs, including the structurally and genetically related phenylalanyl-tRNA synthetase (PheRS), strictly recognize the main chain groups of the substrate. We report here that wild type PylRS can recognize substrates with a variety of main-chain α-groups: α-hydroxyacid, non-α-amino-carboxylic acid, N_α-methyl-amino acid, and d-amino acid, each with the same side chain as that of Boc-lysine. In contrast, PheRS recognizes none of these amino acid analogs. By expressing the wild type PylRS and its cognate tRNA^Pyl in E. coli in the presence of the α-hydroxyacid analog of Boc-lysine (Boc-LysOH), the amber codon (UAG) was recoded successfully as Boc-LysOH, and thus an ester bond was site-specifically incorporated into a protein molecule. This PylRS-tRNA^Pyl pair is expected to expand the backbone diversity of protein molecules produced by both in vivo and in vitro ribosomal translation.     

Distinct genetic code expansion strategies for selenocysteine and pyrrolysine are reflected in different aminoacyl-tRNA formation systems

Selenocysteine and pyrrolysine, known as the 21st and 22nd amino acids, are directly inserted into growing polypeptides during translation. Selenocysteine is synthesized via a tRNA-dependent pathway and decodes UGA (opal) codons. The incorporation of selenocysteine requires the concerted action of specific RNA and protein elements. In contrast, pyrrolysine is ligated directly to tRNA^Pyl and inserted into proteins in response to UAG (amber) codons without the need for complex re-coding machinery. Here we review the latest updates on the structure and mechanisms of molecules involved in Sec-tRNA^Sec and Pyl-tRNA^Pyl formation as well as the distribution of the Pyl-decoding trait.     

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA^Pyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA^Pyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.     

Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA^Pyl pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA^Pyl in Chinese hamster ovary cells. When tRNA^Pyl was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA^Pyl pair facilitated the most efficient incorporation of a pyrrolysine analog, N^ε-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA^Pyl system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N^ε-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N^ε-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.     

Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase

Pyrrolysine, a lysine derivative with a bulky pyrroline ring, is the “22nd” genetically encoded amino acid. In the present study, the carboxy-terminal catalytic fragment of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) was analyzed by X-ray crystallography and site-directed mutagenesis. The catalytic fragment ligated tRNA^Pyl with pyrrolysine nearly as efficiently as the full-length PylRS. We determined the crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)-bound, and AMPPNP/pyrrolysine-bound forms, and compared them with the previously-reported PylRS structures. The ordering loop and the motif-2 loop undergo conformational changes from the “open” states to the “closed” states upon AMPPNP binding. On the other hand, the β7-β8 hairpin exhibits multiple conformational states, the open, intermediate (β7-open/β8-open and β7-closed/β8-open), and closed states, which are not induced upon substrate binding. The PylRS structures with a docked tRNA suggest that the active-site pocket can accommodate the CCA terminus of tRNA when the motif-2 loop is in the closed state and the β7-β8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the β7-β8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNA^Pyl. Moreover, a structure-based mutational analysis revealed that hydrophobic residues in the amino acid-binding tunnel are important for accommodating the pyrrolysine side chain and that Asn346 is essential for anchoring the side-chain carbonyl and α-amino groups of pyrrolysine. In addition, a docking model of PylRS with tRNA was constructed based on the aspartyl-tRNA synthetase/tRNA structure, and was confirmed by a mutational analysis.     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNAPyl Pairs in the Escherichia coli BL21(DE3) Cell Strain

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA^Pyl pairs and cross recognition between nonsense codons and various tRNA^Pyl anticodons in the Escherichia coli BL21(DE3) cell strain are reported. is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, N^ε-tert-butoxycarbonyl-l-lysine (BocK). Similar to, is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS- pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS- pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNA^Pyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNA^Pyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNA^Pyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNA^Pyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNA^Pyl2 charging by PylRS2 is defined by the enzyme''s shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNA^Pyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.     

Combination of the Loss of cmnmU34 with the Lack of sU34 Modifications of tRNA, tRNA, and tRNA Altered Mitochondrial Biogenesis and Respiration

Yeast Saccharomyces cerevisiae MTO2, MTO1, and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of carboxymethylaminomethyl (cmnm)⁵s²U₃₄ in mitochondrial tRNA^Lys, tRNA^Glu, and tRNA^Gln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm⁵ group (cmnm⁵U₃₄), while Mto2p is responsible for the 2-thiouridylation (s²U₃₄) of these tRNAs. Previous studies showed that partial modifications at U₃₄ in mitochondrial tRNA enabled mto1, mto2, and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1, and MSS1 genes by using the mto2, mto1, and mss1 single, double, and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol but not on medium containing glucose. Interestingly, there were no detectable levels of nine tRNAs including tRNA^Lys, tRNA^Glu, and tRNA^Gln in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1, and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA and 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln, caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm⁵U₃₄ by the mto1 or mss1 mutation. The complete loss of modifications at U₃₄ of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA, and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory-deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U₃₄ of tRNA^Lys, tRNA^Glu, and tRNA^Gln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation, and respiratory function.     

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg and NADPH Binding as Revealed by Two Crystal Structures

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions—alkyl migration and reduction—and requires Mg²⁺ and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn²⁺-(phospho)ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg²⁺-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI-Mg²⁺ and rice KARI-Mg²⁺-NADPH complexes to 1.55 Å and 2.80 Å resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15° relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ∼ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg²⁺ complex are disordered. In the rice KARI-Mg²⁺-NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release.     

Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA^Pyl to create tRNA^Pyl-opt with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N^ϵ-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA^Pyl-opt facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA^Pyl-opt with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA^Pyl-opt had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA^Pyl-opt should be an excellent replacement of wild-type tRNA^Pyl for future ncAA incorporation by PylRS enzymes.     

Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs

It was recently shown that the structure of the fluorophore attached to the acyl chain of phosphatidylcholine analogs determines their mechanism of transport across the plasma membrane of yeast cells (Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight into the physical properties of these fluorescent phosphatidylcholine (PC) analogs, the rate and mechanism of their intervesicular transport was determined. The rate of spontaneous exchange was measured for PC analogs containing either NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene), Bodipy 530 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene), or Bodipy 581 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene) attached to a five or six carbon acyl chain in the sn-2 position. The rate of transfer between phospholipid vesicles was measured by monitoring the increase in fluorescence as the analogs transferred from donor vesicles containing self-quenching concentrations to unlabeled acceptor vesicles. Kinetic analysis indicated that the transfer of each analog occurred by diffusion through the water phase as opposed to transfer during vesicle collisions. The vesicle-to-monomer dissociation rate constants differed by over four orders of magnitude: NBD-PC (k_dis = 0.115 s^− 1; t_1/2 = 6.03 s); Bodipy FL-PC (k_dis = 5.2 × 10^− 4; t_1/2 = 22.2 min); Bodipy 530-PC (k_dis = 1.52 × 10^− 5; t_1/2 = 12.6 h); and Bodipy 581-PC (k_dis = 5.9 × 10^− 6; t_1/2 = 32.6 h). The large differences in spontaneous rates of transfer through the water measured for these four fluorescent PC analogs reflect their hydrophobicity and may account for their recognition by different mechanisms of transport across the plasma membrane of yeast.     

Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2₁ at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.