M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.

The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M.phi 3TI and M.rho 11sI, which were previously characterized, for the identical monospecific C5-MTases M.phi 3TII and M.rho 11sII. These enzymes modify the C to TCGA sites, a novel target specificity among C5-MTases. The primary sequence of M.phi 3TII (326 amino acids) shows all conserved motifs typical of the building plan of C5-MTases. The degree of relatedness between M.phi 3TII and all other mono- or multispecific C5-MTases ranges from 30-40% amino acid identity. Particularly M.phi 3TII does not show pronounced similarity to M.phi 3TI indicating that both MTase genes were not generated from one another but were acquired independently by the phage. The amino terminal part of the M.phi 3TII (preceding the variable region 'V'), which predominantly constitutes the catalytic domain of the enzyme, exhibits pronounced sequence similarity to the amino termini of a family of A-N6-MTases, which--like M.Taql--recognize the general sequence TNNA. This suggests that recently described similarities in the general three dimensional organization of C5- and A-N6-MTases imply divergent evolution of these enzymes originating from a common molecular ancestor.     

The amino acid sequence of the eukaryotic DNA [N6-adenine]methyltransferase, M.CviBIII, has regions of similarity with the prokaryotic isoschizomer M.TaqI and other DNA [N6-adenine] methyltransferases

The sequences of the genes coding for M.CviBIII (from virus NC-1A which infects a eukaryotic alga) [Narva et al., Nucleic Acids Res. 15 (1987) 9807-9823] and M.TaqI (from the bacterium Thermus aquaticus) [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] have been determined recently. Both enzymes methylate adenine in the sequence TCGA. We have compared the predicted amino acid sequences of these two methyltransferases (MTases), with each other and with ten other N6 A-MTases and find regions of similarity. M.CviBIII and M.TaqI were most closely related followed by M.PaeR7, whose recognition sequence (CTCGAG) contains the M.TaqI/M.CviBIII recognition sequence TCGA, and M.PstI, whose recognition sequence is CTGCAG. All of the N6-MTases contain the sequence Asp/Asn-Pro-Pro-Tyr (B-P-P-Y) referred to by Hattman et al. [J. Bacteriol. 164 (1985) 932-937] as region IV. The predicted secondary structure of this region forms a finger-like structure ('beta finger') containing a beta-pleated sheet (...XXXB), two beta-turns (P-P) followed by another beta-pleated sheet [Y/FXXX...].     

M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed.     

The amino acid sequence of the CCGG recognizing DNA methyltransferase M.BsuFI: implications for the analysis of sequence recognition by cytosine DNA methyltransferases.

The Bacillus subtilis FI DNA methyltransferase (M.BsuFI) modifies the outer cytosine of the DNA sequence CCGG, causing resistance against R.BsuFI and R.MspI restriction. The M.BsuFI gene was cloned and expressed in B.subtilis and Escherichia coli. As derived from the nucleotide sequence, the M.BsuFI protein has 409 amino acids, corresponding to a molecular mass of 46,918 daltons. Including these data we have compared the nucleotide and amino acid sequences of different CCGG recognizing enzymes. These analyses showed that M.BsuFI is highly related to two other CCGG specific methyltransferases, M.MspI and M.HpaII, which were isolated from Gram-negative bacteria. Between M.BsuFI and M.MspI the sequence similarity is particularly significant in a region, which has been postulated to contain the target recognition domains (TRDs) of cytosine-specific DNA methyltransferases. Apparently M.BsuFI and M.MspI, derived from phylogenetic distant organisms, use highly conserved structural elements for the recognition of the CCGG target sequence. In contrast the very same region of M.HpaII is quite different from those of M.BsuFI and M.MspI. We attribute this difference to the different targeting of methylation within the sequence CCGG, where M.HpaII methylates the inner, M.BsuFI/M.MspI the outer cytosine. Also the CCGG recognizing TRD of the multispecific B.subtilis phage SPR Mtase is distinct from that of the host enzyme, possibly indicating different requirements for TRDs operative in mono- and multispecific enzymes.     

Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family

Transfer RNA (Gm18) methyltransferase (TrmH (SpoU)) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 2'-OH of guanosine 18 in tRNA. This enzyme is a member of the SpoU family of RNA methyltransferases. Recent computational researches have shown that three amino acid sequence motifs are conserved among the SpoU members. Recently, we determined the crystal structures of the apoand AdoMet bound forms of TrmH (Nureki, O., Watanabe, K., Fukai, S., Ishii, R., Endo, Y., Hori, H., and Yokoyama, S. (2004) Structure 12, 593-602). Furthermore, we clarified the AdoMet binding site and proposed the catalytic mechanism. Since the functions of the conserved amino acid residues in the motifs remain unknown, here we have prepared 17 mutants of TrmH and carried out various biochemical studies, including determination of the kinetic parameters for both AdoMet and tRNA, S-adenosyl-l-homocysteine affinity chromatography, gel mobility shift assay, CD spectroscopy, and analytical gel filtration. Our results show that Asn(35), Arg(41), Glu(124), and Asn(152) are involved in binding tRNA and that the Asn(35) residue is involved in the release of S-adenosyl-l-homocysteine. Several residues of TrmH are important for stability of the enzyme. Taken together, our biochemical studies reinforce the previously proposed catalytic mechanism. We also discuss amino acid substitutions in general within the SPOUT superfamily of methyltransferases.     

A new DNA methyltransferase M.BstC8I forms 5'-G(M5C)NNGC-3'. Studying of restriction endonuclease sensitivity to M.BstC8I-methylation

Bacillus stearothermophilus C8 was grown up on the Luria agar at 37 degrees C. A new DNA-methylase was determined in cellular lysate. The methylation of the DNAs of bacteriophages lambda and T7 in the region of 5'-G(m5C)NNGC-3' blocked the activity of BstC8I. Specificity of M.BstC8I was analyzed on methylated lambda DNA. For this purpose, we used computer modeling and the data on the sensitivity of restrictases BstC8I, BsuRI, AjnI, and PvuII to methylation. The sensitivity of some restrictases to new methylation was studied. The results may be used for DNA methylation studying.     

Single amino acid changes that alter the DNA sequence specificity of the DNA-[N6-adenine] methyltransferase (Dam) of bacteriophage T4.

Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam) which recognizes primarily the sequence GATC in both cytosine- and hydroxymethylcytosine-containing DNA. Hypermethylating mutants, damh, exhibit a relaxation in sequence specificity, that is, they are readily able to methylate non-canonical sites. We have determined that the damh mutation produces a single amino acid change (Pro126 to Ser126) in a region of homology (III) shared by three DNA-adenine methyltransferases; viz, T4 Dam, Escherichia coli Dam, and the DpnII modification enzyme of Streptococcus pneumoniae. We also describe another mutant, damc, which methylates GATC in cytosine-containing DNA, but not in hydroxymethylcytosine-containing DNA. This mutation also alters a single amino acid (Phe127 to Val127). These results implicate homology region III as a domain involved in DNA sequence recognition. The effect of several different amino acids at residue 126 was examined by creating a polypeptide chain terminating codon at that position and comparing the methylation capability of partially purified enzymes produced in the presence of various suppressors. No enzyme activity is detected when phenylalanine, glutamic acid, or histidine is inserted at position 126. However, insertion of alanine, cysteine, or glycine at residue 126 produces enzymatic activity similar to Damh.     

γ-Purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm

Two homologous sulfur-rich basic polypeptides form wheat endosperm, so-called γ₁-purothionin and γ₂-purothionin, are described. Purification involves extraction with volatile solvents and ammonium bicarbonate fractionation followed by reversed-phase high-performance liquid chromatography. The complete primary structure of these two polypeptides has been determined by automatic degradation of the intact, S-carboxymethylated γ-purothionins and peptides obtained by enzymatic cleavage. γ₁-Purothionin and γ₂-purothionin consist of 47 amino acids with an assessed molecular weight of 5239 and 5151 Da, respectively and 8 cysteines organized in 4 disulfide bridges. They present a high degree of homology among themselves (89% of identity) and are the first two thionin-like polypeptides, so-called y-thionins, described from wheat endosperm.     

Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases.

A large portion of the sequences of type II DNA-(cytosine-C5)-methyltransferases (C5-MTases) represent highly conserved blocks of amino acids. General steps in the methylation reaction performed by C5-MTases have been found to be mediated by some of these domains. C5-MTases carry, in addition at the same relative location, a region variable in size and amino acid composition, part of which is associated with the capacity of each C5-MTase to recognize its characteristic target. Individual target-recognizing domains (TRDs) for the targets CCGG (M), CC(A/T)GG (E), GGCC (H), GCNGC (F) and G(G/A/T)GC(C/A/T)C (B) could be identified in the C-terminal part of the variable region of multispecific C5-MTases. With experiments reported here, we have established the organization of the variable regions of the multispecific MTases M.SPRI, M.phi3TI, M.H2I and M.rho 11SI at the resolution of individual amino acids. These regions comprise 204, 175, 268 and 268 amino acids, respectively. All variable regions are bipartite. They contain at their N-terminal side a very similar sequence of 71 amino acids. The integrity of this sequence must be assured to provide enzyme activity. Bracketed by 6-10 'linker' amino acids, they have, depending on the enzyme studied, towards their C-terminal end ensembles of individual TRDs of 38 (M), 39 (E), 40 (H), 44 (F) and 54 (B) amino acids. TRDs of different enzymes with equal specificity have the same size. TRDs do not overlap but are either separated by linker amino acids or abut each other.     

Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog

The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (Bra/LIV) of the ABC family

Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra(Rl)). Characterization of the solute specificity of Bra(Rl) shows it to be the second general amino acid permease of R. leguminosarum. Although Bra(Rl) has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (L-glutamate, L-arginine, and L-histidine), in addition to neutral amino acids (L-alanine and L-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be alpha-amino acids. Consistent with this, Bra(Rl) is the first ABC transporter to be shown to transport gamma-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by Bra(Rl) does not appear to be stereospecific as D amino acids cause significant inhibition of uptake of L-glutamate and L-leucine. Unlike all other solutes tested, L-alanine uptake is not dependent on solute binding protein BraC(Rl). Therefore, a second, unidentified solute binding protein may interact with the BraDEFG(Rl) membrane complex during L-alanine uptake. Overall, the data indicate that Bra(Rl) is a general amino acid permease of the HAAT family. Furthermore, Bra(Rl) has the broadest solute specificity of any characterized bacterial amino acid transporter.     

The amino acid sequence of a human immunoglobulin G3m(g) pFc' fragment.

Amino acid sequence analysis of the pFc' fragment obtained by pepsin digestion of an IgG3; G3m(g) human myeloma protein HER shows it to consist of 112 residues. It starts at position 334 (gamma1 numbering), contains eight residues of the Cgamma2 region, and the whole Cgamma3 domain. Comparison with the sequence of gamma1 shows five differences including an extra Met at 397. Each is accountable by a single base substitution. The sequence is identical to that of a G3m(b0) molecule except for the previously noted allotype related Tyr/Phe exchange at position 436. The high degree of homology (95%) among gamma-chain subclasses suggests a recent diversification.     

Purification of chitinolytic protein from Rehmannia glutinosa showing N-terminal amino acid sequence similarity to thaumatin-like proteins.

We have purified a 21-kDa protein, designated as P1, from Rehmannia glutinosa to homogeneity by ammonium sulfate precipitation, anion exchange chromatography, hydrophobic interaction chromatography, and preparative native PAGE. The purified P1 had chitin degradation activity. The N-terminal amino acid sequence of P1 indicated that it is very similar to those of thaumatin and other reported thaumatin-like proteins.     

Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6)

Two protein families that represent major components of essential amino acid transport in insects have been identified. They are annotated as the SLC6 and SLC7 families of transporters according to phylogenetic proximity to characterized amino acid transporters (HUGO nomenclature). Members of these families have been identified as important apical and basolateral parts of transepithelial essential amino acid absorption in the metazoan alimentary canal. Synergistically, they play critical physiological roles as essential substrate providers to diverse metabolic processes, including generic protein synthesis. This review briefly clarifies the requirements for amino acid transport and a variety of amino acid transport mechanisms, including the aforementioned families. Further it focuses on the large group of Nutrient Amino acid Transporters (NATs), which comprise a recently identified subfamily of the Neurotransmitter Sodium Symporter family (NSS or SLC6). The first insect NAT, cloned from the caterpillar gut, has a broad substrate spectrum similar to mammalian B(0) transporters. Several new NAT-SLC6 members have been characterized in an effort to explore mechanisms for the essential amino acid absorption in model dipteran insects. The identification and functional characterization of new B(0)-like and narrow specificity transporters of essential amino acids in fruit fly and mosquitoes leads to a fundamentally important insight: that NATs evolved and act together as the integrated active core of a transport network that mediates active alimentary absorption and systemic distribution of essential amino acids. This role of NATs is projected from the most primitive prokaryotes to the most complex metazoan organisms, and represents an interesting platform for unraveling the molecular evolution of amino acid transport and modeling amino acid transport disorders. The comparative study of NATs elucidates important adaptive differences between essential amino acid transportomes of invertebrate and vertebrate organisms, outlining a new possibility for selective targeting of essential amino acid absorption mechanisms to control medically and economically important arthropods and other invertebrate organisms.