Evolution of the biosynthesis of the branched-chain amino acids

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from -ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.     

Coat proteins of strains of two RNA viruses: comparison of their amino acid sequences

Summary The amino acid sequences of four strains of tobacco mosaic virus isolated in different parts of the world are compared. The differences between the strains are discussed with respect to special proteinchemical features (such as beginning of the chain, deletion of amino acids, number of different amino acids, sizes and distribution of regions with invariable amino acids) and with respect to the possibility of deducing the most probable nucleotide sequence for the coat protein cistron of tobacco mosaic virus.The complete amino acid sequences of the two RNA bacteriophage strains fr and f₂ are compared. According to their coat proteins three groups of phages can be formed: 1) MS 2, f₂ M 12 and R 17, 2) fr and 3) Q.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Crossassociation: A method of comparing protein sequences

Crossassociation is a computer method of comparing protein sequences. It can help detect amino acid matches, deletions, insertions, and other similarities which would be hard to detect by eye. The method is to slide the sequences past each other one step at a time and to count the number of amino acids that match. At each overlap position, the program prints the percentage match and statistical significance measures of the matching. The null hypothesis for significance is the random arrangement of amino acids in the proportions found in the sequences under study. For most protein pairs, the expected proportion of matches is about 1/14. The method includes computation of three overall similarity measures between sequences which should have use in both evolutionary and taxonomic studies. The use of the method has been tested with actual and hypothetical sequences. Problems of recovering evolutionary relationships by this and related methods are discussed.     

Dipeptide frequencies in proteins and the CpG deficiency in vertebrate DNA

Summary Analysis of vertebrate protein sequences totalling 4040 residues shows that amino acids with a high proportion of codons ending in C occur with significantly reduced frequency before amino acids whose codons start with G. This effect is not shown by control bacterial protein sequences. The consequent implication of shortage of XXC. GXX codon pairs in vertebrate messenger RNA is discussed in relation to the extreme rarity of the base doublet CpG in vertebrate DNA.     

Some analogies between prebiotic thermal conversions of glycine and metabolic pathways

The reaction schemes suggested earlier for thermal transformation of glycine into amino acids and carboxylic acids are considered in detail. Close analogy with some wide-spread biochemical reactions of amino acids is observed. The pathway suggested has some common stages with the tricarboxylic acid cycle and other metabolic processes. The possible role of -imino or -keto acids as prebiological analogs of pyridoxal-phosphate-containing enzymes is discussed. The thermal transformations of glycine under primitive Earth conditions could be considered as evolutionary precursors of some present-day metabolic pathways.     

Transformation of some hydroxy amino acids to other amino acids

It has been observed that -hydroxy--amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give -hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of -hydroxy--amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.     

The genes coding for the L,M and cytochrome subunits of the photosynthetic reaction center from the thermophilic purple sulfur bacterium t Chromatium tepidum

The complete nucleotide sequences of the genes coding for L, M protein subunits and part of cytochrome subunit of the photosynthetic reaction center were determined for the thermophilic purple sulfur bacterium t Chromatium tepidum (t Chr. tepidum) which belongs to the subclass. The DNA fragments with 860 bp and 1900 bp were amplified by the Polymerase Chain Reaction (PCR) with the primers designed on the basis of amino acid sequences according to chemical sequence analysis of the proteins. The deduced amino acid sequences of these genes showed a significantly high degree of homology with those from purple non-sulfur bacteria. The L subunit consisted of 280 amino acids and had a molecular mass of 31,393. The M subunit consisted of 324 amino acids and had a molecular mass of 36,299. The aligned sequences of the L subunits of other purple bacterial reaction center polypeptides, showed the insertion of 8 amino acids in t Chr. tepidum in the connection of the first and second membrane-spanning helices different from those of purple non-sulfur bacteria. The aligned sequences of the L, M and cytochrome subunits were compared with other species and discussed in terms of phylogenetic trees.     

Multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles

Summary Amino acids enter rabbit jejunal brush border membrane vesicles via three major transport systems: (1) simple passive diffusion; (2) Na-independent carriers; and (3) Na-dependent carriers. The passive permeability sequence of amino acids is very similar to that observed in other studies involving natural and artificial membranes. Based on uptake kinetics and cross-inhibition profiles, at least two Na-independent and three Na-dependent carrier-mediated pathways exist. One Na-independent pathway, similar to the classical L system, favors neutral amino acids, while the other pathway favors dibasic amino acids such as lysine. One Na-dependent pathway primarily serves neutrall-amino acids including 2-amino-2-norbornanecarboxylic acid hemihydrate (BCH), but not -alanine or -methylaminoisobutyric acid (MeAIB). Another Na-dependent route favors phenylalanine and methionine, while the third pathway is selective for imino acids and MeAIB. Li is unable to substitute for Na in these systems. Cross-inhibition profiles indicated that none of the Na-dependent systems conform to classical A or ACS paradigms. Other notable features of jejunal brush border vesicles include (1) no -alanine carrier, and (2) no major proline/glycine interactions.     

Molecular cloning and sequence analysis of an endo-inulinase gene from Arthrobacter sp.

A 3.0-kb DNA fragment containing an endo-inulinase gene was cloned from Arthrobacter sp. S37. It contained a single open reading frame of 2439 bp, encoding a polypeptide composed of signal peptide of 53 amino acids and mature protein of 759 amino acids. From the comparison with amino acids sequences of fructan hydrolases and invertase, five highly conserved regions including the -fructosidase motif were found. The sequence of the endo-inulinase had the identity in the range of 13.3% to 16.0%.     

Evaluation of two new coupling agents for incorporation of α,α-dialkylamino acids, such as α-methylalanine, in solid-phase peptide synthesis

Summary The introduction of solid-phase peptide synthesis (SPPS) has greatly facilitated the preparation of peptides containing proteinaceous amino acids. Less common, sterically hindered ,-dialkylamino acids, such as -methylalanine (MeA, aminoisobutyric acid, Aib), have proven a synthetic challenge for incorporation by this approach, especially when present in contiguous sequences. Solution protocols, utilizing highly reactive intermediates such as oxazalones, are generally used during the preparation of peptaibol antibiotics such as alamethicin, emerimicin, etc. which contain such contiguous sequences. Two recently developed coupling strategies (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HATU, and Fmoc-protected amino acid fluorides) allow peptides comprising contiguous sequences of ,-dialkylamino acids to be prepared using SPPS. The present study evaluates the relative merits of these two methods on a set of difficult peptides containing oligo-MeA sequences.     

Experimental retracement of the origins of a protocell

Although Oparin used coacervate droplets from two or more types of polymer to model the first cell, he hypothesized homacervation from protein, consistent with Pasteur and Darwin. Herrera made two amino acids and numerous cell-like structures (sulfobes) in the laboratory, which probably arose from intermediate polymers. Our experiments have conformed with a homoacervation of thermal proteinoid, in which amino acid sequences are determined by the reacting amino acids themselves. All proteinoids that have been tested assemble themselves alone in water to protocells. The protocells have characteristics of life defined by Webster's Dictionary: metabolism, growth, reproduction and response to stimuli in the environment. The protocells are able also to evolve to more modern cells including the initiation of a nucleic acid coding system.Principal spinoffs from the results are revised evolutionary theory, models for protoneurons and networks thereof, and numerous industrial applications of thermal polyamino acids. Life itself has thus been reaffirmed to be rooted in protein, not in DNA nor RNA, which are however crucial to inheritance in modern life as instruction manual (Kornberg).Recognition of the advances have been considerably delayed by the deeply held assumption that life began by chance from random polymerization of amino acids, in contrast to the experimental findings. The concepts of DNA/RNA-first and protein-first are reconciled by a rise-and-fall progression as often seen in biochemical and biological evolution.The fact that amino acids order themselves explains in turn that thermal copolyamino acids are finding numerous applications. The entire sequence of processes in the proteinoid origins theory is now seen to be highly deterministic, in close accord with Einstein.     

Proteolytic Processing of Oligopeptides Containing the Target Sequences by the Recombinant Tobacco Vein Mottling Virus NIa Proteinase

Tobacco vein mottling virus (TVMV) belongs to the potyviridae that consists of about 200 plant viruses. Potyviruses have RNA genomes of approximately 10,000 bases from which a single polyprotein is expressed from each virus upon infection. The NIa proteinase is known to process the polyprotein at seven distinct junctions between proteins. Kinetic constants were determined for the reactions of the recombinant TVMV NIa protease (27 kDa) with synthetic oligopeptides containing the sequences for the cleavage sites. For optimum activity, the substrate needs to have six amino acids (P6–P1) in the amino region and four (P1–P4) in the carboxy region, including four conserved amino acids (V-R-F-Q) in P4P1 positions. Mutation of any of four conserved amino acids to Gly made the substrate inert to the enzyme. Among the substrates, the oligopeptides containing the sequences for junctions, P3–6K1, NIa (VPg-Pro), and NIa-NIb were not processed by the NIa protease. Those junctions have Glu at P3, Glu at P1, and Thr at P2. The implications of high substrate specificity and size dependence in polyprotein processing and viral replication are discussed.     

A human metallothionein pseudogene containing AG/CT repetitive elements

Summary A lambda phage recombinant clone, 25 S, which contains a 15.5-kb EcoRI human genomic DNA fragment, has been characterized. Restriction mapping and Southern blot hybridization indicated a 3.0-kb HindIII fragment containing metallothionein (MT)-like sequences. Several interesting features were found upon comparison of this nucleotide sequence with that of other human MT genes: (1) sequences representing the 5 regulatory region, the 5 untranslated region, and the first exon are not contained in the 3.0-kb HindIII fragment; (2) the coding sequence of the second exon (amino acids 10–31 encoding a portion of the -domain of the MT protein) has 11 amino acid changes out of a total of 21, whereas, the third exon (amino acids 32–61, representing the complete -domain of the MT protein) has only 4 amino acid substitutions; however, all cysteine residues are conserved; (3) this MT-like gene retains intron sequences and processing signals; (4) Southern blot analysis of human genomic DNA indicated this MT-like gene is located on a 10.5-kb EcoRI genomic DNA fragment; and (5) unusual AG/CT-rich repetitive elements are located within the second intron and upstream of the second exon of this MT-like gene. This gene is not expressed in response to metal induction in two human cell lines, as shown by northern blot analyses. Based on these observations, this MT-like gene represents a unique nonprocessed pseudogene of the human MT multigene family.     

The small protein floodgates are opening; now the functional analysis begins

Aside from a few serendipitous discoveries, small proteins of less than 50 amino acids in bacteria and 100 amino acids in eukaryotes were largely ignored due to challenges in their genetic and biochemical detection. However, with the ever-increasing availability of completed genome sequences and deep sequencing, which allows analysis of genome-wide ribosome occupancy, hundreds of small proteins are now being identified. This brings to the forefront the challenges and opportunities associated with the characterization of these proteins.See research article: http://www.biomedcentral.com/1471-2164/15/946.     

A ple?otropic acid phosphatase-deficient mutant of Escherichia coli shows premature termination in the dsbA gene. Use of dsbA :: phoA furions to localize a structurally important domain in DsbA

An Arabidopsis thaliana cDNA library was used to complement Saccharomyces cerevisiae pyrimidine auxotrophic mutants. Mutants in all but one (carbamylphosphate synthetase) of the six steps in the de novo pyrimidine biosynthetic pathway could be complemented. We report here the cloning, sequencing and computer analysis of two cDNAs encoding the aspartate transcarbamylase (ATCase; EC 2.1.3.2) and orotate phosphoribosyltransferase-orotidine-5-phosphate decarboxylase (OPRTase-OMP-decase; EC 2.4.2.10, EC 4.1.1.23) enzymes. These results confirm the presence in A. thaliana of a bifunctional gene whose product catalyses the last two steps of the pyrimidine biosynthetic pathway, as previously suggested by biochemical studies. The ATCase encoding cDNA sequence (PYRB gene) shows an open reading frame (ORF) of 1173 by coding for 390 amino acids. The cDNA encoding OPRTase-OMPdecase (PYRE-F gene) shows an ORF of 1431 by coding for 476 amino acids. Computer analysis of the deduced amino acid sequences of both cDNAs shows the expected high similarity with the ATCase, ornithine transcarbamylase (OTCase; EC 2.1.3.3), OPRTase and OMPdecase families. This heterospecific cloning approach increases our understanding of the genetic organization and interspecific functional conservation of the pyrimidine biosynthetic pathway and underlines its usefulness as a model for evolutionary studies.     

Formation of amino acids on heating glycine with alumina

The conversion of glycine into amino acids on heating at 240°C with basic manganous carbonate and alumina is investigated. Alanine, -aminobutyric acid, norvaline, norleucine, sarcosine, N-ethylglycine, N-methylalanine, N-ethylalanine, aspartic acid and glutamic acid are identified among the products of the reaction. Paper chromatography, ion-exchange chromatography and nuclear magnetic resonance are used for the analysis. A scheme for the observed transformations is presented and it is suggested that it may have been a pathway for the synthesis of amino acids from glycine under primitive Earth conditions.     

Cloning and nucleotide sequence of β-mannanase and cellulase genes from Bacillus sp. 5H

The two genes for -mannanase and cellulase of Bacillus sp. 5H have been cloned in Escherichia coli JM 109 by a shotgun method, though the cellulase gene was not expressed in Bacillus sp. 5H. The nucleotide sequences of the -mannanase gene and the cellulase gene revealed open reading frames of 1,086 and 1,503 base pairs, respectively, coding for a proteins of M_r 40,803 Da (-mannanase) and 55,420 Da (cellulase). The deduced primary structure of -mannanase comprised 362 amino acids which had a mature protein of 336 amino acids and a signal peptide of 26 amino acids and that of cellulase comprised 501 amino acid residues.     

Possible palindromes in immunoglobulin heavy-chain genes: Their role in membrane attachment

An analysis of mRNA structures as deduced from the amino acid sequences of immunoglobulin heavy chains reveals possible base-paired regions within the sequences coding for the 20 C-terminal amino acids of the human and chains. The two regions are similar in structure and contain a palindrome which might serve as an enzyme recognition site. Although other base-paired regions can be predicted in the remainder of the constant regions of these heavy chains, they have no common features. These regions and the palindromes within them may be involved in the regulation of membrane and serum immunoglobulin synthesis. Two possible mechanisms for this are proposed.