Kinetic mechanism of the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA)

The bacterial enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the unprecedented transfer and isomerization of the ribosyl moiety of S-adenosylmethionine (AdoMet) to a modified tRNA nucleoside in the biosynthesis of the hypermodified nucleoside queuosine. The complexity of this reaction makes it a compelling problem in fundamental mechanistic enzymology, and as part of our mechanistic studies of the QueA-catalyzed reaction, we report here the elucidation of the steady-state kinetic mechanism. Bi-substrate kinetic analysis gave initial velocity patterns indicating a sequential mechanism, and provided the following kinetic constants: K (M)(tRNA)= 1.9 +/- 0.7 microM and K (M)(AdoMet)= 98 +/- 5.0 microM. Dead-end inhibition studies with the substrate analogues S-adenosylhomocysteine and sinefungin gave competitive inhibition patterns against AdoMet and noncompetitive patterns against preQ(1)-tRNA(Tyr), with K(i) values of 133 +/- 18 and 4.6 +/- 0.5 microM for sinefungin and S-adenosylhomocysteine, respectively. Product inhibition by adenine was noncompetitive against both substrates under conditions with a subsaturating cosubstrate concentration and uncompetitive against preQ(1)-tRNA(Tyr) when AdoMet was saturating. Inhibition by the tRNA product (oQ-tRNA(Tyr)) was competitive and noncompetitive against the substrates preQ(1)-tRNA(Tyr) and AdoMet, respectively. Inhibition by methionine was uncompetitive versus preQ(1)-tRNA(Tyr), but noncompetitive against AdoMet. However, when methionine inhibition was investigated at high AdoMet concentrations, the pattern was uncompetitive. Taken together, the data are consistent with a fully ordered sequential bi-ter kinetic mechanism in which preQ(1)-tRNA(Tyr) binds first followed by AdoMet, with product release in the order adenine, methionine, and oQ-tRNA. The chemical mechanism that we previously proposed for the QueA-catalyzed reaction [Daoud Kinzie, S., Thern, B., and Iwata-Reuyl, D. (2000) Org. Lett. 2, 1307-1310] is consistent with the constraints imposed by the kinetic mechanism determined here, and we suggest that the magnitude of the inhibition constants for the dead-end inhibitors may provide insight into the catalytic strategy employed by the enzyme.     

Crystal structure of Bacillus subtilis S-adenosylmethionine:tRNA ribosyltransferase-isomerase

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. It is unprecedented in nature as it uses the cofactor S-adenosylmethionine as the donor of a ribosyl group. We have determined the crystal structure of Bacillus subtilis QueA at a resolution of 2.9A. The structure reveals two domains representing a 6-stranded beta-barrel and an alpha beta alpha-sandwich, respectively. All amino acid residues invariant in the QueA enzymes of known sequence cluster at the interface of the two domains indicating the localization of the substrate binding region and active center. Comparison of the B. subtilis QueA structure with the structure of QueA from Thermotoga maritima suggests a high domain flexibility of this enzyme.     

tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily

TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2′-O modification at ribose 32. Herein, we demonstrate that all six Escherichia coli (Ec) tRNAs with 2′-O-methylated nucleosides at position 32 are substrates of EcTrmJ, and we show that the elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations. Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding. We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes.

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.     

发酵重组Pichia pastoris生产腺苷甲硫氨酸的研究

在5L发酵罐中对高产S腺苷甲硫氨酸的重组Pichia pastoris发酵进行了研究。重组菌在pH5．0生长,然后调为pH6．0积累腺苷甲硫氨酸,在30℃、溶氧5％及流加甲硫氨酸和尿素的条件下培养82h后,产量达4．3g／L。     

A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase

The modified nucleoside 1-methyladenosine (m¹A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m¹A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m¹A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m¹I57). In yeast, the formation of m¹A58 is catalysed by the essential tRNA (m¹A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m¹A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m¹A MTases is discussed in the context of different hypotheses of the tree of life.     

Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN.

The gene encoding RNase BN was localized to 88 min on the Escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis. Assay of subclones derived from lambda phage 543 of the Kohara library, which encompasses this region of the chromosome, for elevated RNase BN activity identified o290, a previously reported open reading frame, as the gene encoding RNase BN. Interruption of this gene with a Kan(r) cassette and introduction into the chromosome eliminated cellular RNase BN activity but had no effect on cell growth. On the basis of these data, we suggest that o290 be renamed rbn. Potential homologs of rbn in other organisms also were identified.     

Assay and biochemical characterization of angiotensin-I-converting enzyme in cerebrospinal fluid

A spectrofluorimetric method was adapted for determination of angiotensin-I-converting enzyme (ACE) in untreated native cerebrospinal fluid (CSF). Benzyloxycarbonyl-phenylalanyl-histidyl-leucine was applied as enzyme substrate. The biochemical behavior of ACE of CSF (CACE) was studied. The pH optimum was found to be 8.0 using borax phosphate buffer. The determination of Km was 10.7 +/- 3.3 (SD) mumol/l. ACE could be blocked by 8-hydroxyquinoline (100%) and phenanthroline (80%), but only slight inhibition was observed by teprotide (43%), EDTA (40%) and captopril (31%). The influence of various drugs on CACE was also tested. The different biochemical behavior of CACE compared to serum ACE suggested that an isoenzyme exists in CSF. CACE has been found to be elevated in neuroimmunological and inflammatory disorders of the nervous system compared to levels in healthy controls.     

Structural basis of tRNA modification with CO2 fixation and methylation by wybutosine synthesizing enzyme TYW4

Wybutosine (yW), one of the most complicated modified nucleosides, is found in the anticodon loop of eukaryotic phenylalanine tRNA. This hypermodified nucleoside ensures correct codon recognition by stabilizing codon-anticodon pairings during the decoding process in the ribosome. TYW4 is an S-adenosylmethionine (SAM)-dependent enzyme that catalyzes the final step of yW biosynthesis, methylation and methoxycarbonylation. However, the structural basis for the catalytic mechanism by TYW4, and especially that for the methoxycarbonylation, have remained elusive. Here we report the apo and cofactor-bound crystal structures of yeast TYW4. The structures revealed that the C-terminal domain folds into a β-propeller structure, forming part of the binding pocket for the target nucleoside. A comparison of the apo, SAM-bound, and S-adenosylhomocysteine-bound structures of TYW4 revealed a drastic structural change upon cofactor binding, which may sequester solvent from the catalytic site during the reaction and facilitate product release after the reaction. In conjunction with the functional analysis, our results suggest that TYW4 catalyzes both methylation and methoxycarbonylation at a single catalytic site, and in the latter reaction, the methoxycarbonyl group is formed through the fixation of carbon dioxide.     

Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase. Interference by nucleotides modified with diethyl pyrocarbonate or hydrazine

Treatment of tRNA with diethyl pyrocarbonate or hydrazine prior to incubation with the enzyme ATP/CTP:tRNA nucleotidyltransferase and [alpha-32P]ATP results in exclusion of modified bases from labeled molecules. Purines modified with diethyl pyrocarbonate, which interfere with enzyme recognition, cluster at the corner of the tRNA molecule, where the D- and psi-loops are juxtaposed in all 15 tRNAs used in this study. When the enzyme is isolated from Escherichia coli, few other sites of interference are evident near the 3'-end; when the homologous enzyme from yeast is used, more exclusions are apparent near the 3'-end. Modification of uridines with hydrazine has no effect on interaction with the enzyme, except for one uridine near the 3'-end of tRNA(Gly). Interference of enzyme activity by modified bases can be overcome by longer incubation times or increased concentrations of enzyme.     

组蛋白甲基化修饰酶与早期胚胎发育

早期胚胎发育是胚胎发育中细胞分裂与分化最为活跃的时期,也是合子型基因大规模转录的时期,而此时组蛋白的甲基化修饰也显示出动态学的变化。这一时期,在细胞内外信号的共同调控下,经历着一系列基因的激活与抑制,许多调控机制参与其中的调控。而近年来的研究表示,表观遗传学调控显示越来越重要的作用。组蛋白甲基化修饰是表观遗传学重要调控机制之一,在胚胎的早期发育过程中扮演着重要的角色。就近年来组蛋白甲基化修饰酶在早期胚胎发育过程中的作用与功能做一简要综述。