Aminotransfer Reaction Research Articles

Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics.

Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide core consisting of 5-amino-5-deoxyribofuranose (ADR) attached to 6'- N-alkyl-5'- C-glycyluridine (GlyU). Here, we functionally assign and characterize six enzymes from the muraymycin biosynthetic pathway involved in the core assembly that starts from uridine monophosphate (UMP). The biosynthesis is initiated by Mur16, a nonheme Fe(II)- and α-ketoglutarate-dependent dioxygenase, followed by four transferase enzymes: Mur17, a pyridoxal-5'-phosphate (PLP)-dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and downstream enzymes can also process 2'-deoxy-UMP to generate a 2-deoxy-ADR, which is consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers l-Tyr as the amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the ADR precursor consistent with the nucleotidyltransfer reaction occurring after the Mur20-catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5' S,6' S)-GlyU, suggesting that ribosyltransfer occurs prior to N-alkylation of GlyU. Finally, a one-pot, six-enzyme reaction was utilized to generate the ADR-GlyU disaccharide core starting from UMP.

Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast

Compared to (S)-selective amine transaminase ((S)-AT), the (R)-selective counterpart ((R)-AT) has been less studied. As such, a simplified "Motif Sequence Blast" search (Höhne et al. Nat Chem Biol 6:807-813, 2010) was carried out to identify new (R)-ATs from the protein databases. The combined conserved sequence motifs of (R)-ATs based on the previous in silico method of predicting (R)-selective amine transaminase were used as the template sequence for BLASTP search at default settings in NCBI, and six candidate sequences were identified. These putative (R)-AT genes were synthesized and overexpressed in Escherichia coli. Among them, five new (R)-ATs were expressed as soluble protein and showed unusual substrate specificity and high stereoselectivity. Furthermore, several unnatural amino acids, such as D-alanine, D-2-aminobutyric acid, and D-norvaline, were synthesized via the (R)-AT-catalyzed amino transfer reaction to the corresponding keto acids. Optically pure (S)-amines were also obtained by kinetic resolution of racemic amines catalyzed with these new (R)-ATs. Therefore, the Motif Sequence Blast search offers a quick and effective method for in silico identification of new (R)-ATs, and the newly identified (R)-ATs are attractive additions to the toolbox of (R)-ATs for further study and industrial application.

ChemInform Abstract: An Indenone Synthesis Involving a New Aminotransfer Reaction and Its Application to Dibenzopentalene Synthesis.

AbstractThe three‐step procedure to synthesize indenone derivatives (III) tolerates several functional groups.

An Indenone Synthesis Involving a New Aminotransfer Reaction and Its Application to Dibenzopentalene Synthesis

Abstracto‐(Phenylethynyl)phenyllithiums, generated by treatment of o‐(phenylethynyl)bromobenzenes with tBuLi, were quenched with N,N‐dimethylcarboamides to afford various 2‐phenylindenone derivatives in moderate to good yields upon acidic workup. The choice of lithiation reagents was critical. When n‐butyllithium was used the yields were reduced. The N‐alkyl groups in the carboamides were also important: quenching with N,N‐diethylbenzamides also afforded 2,3‐diphenylindenones as the sole isolated products, but quenching with N‐benzoylpiperidine led to 2‐benzoyltolane in good yield. Control experiments revealed that the reactions proceed through a multi‐step reaction sequence involving a novel intramolecular transamination to the α‐acetylenic carbon of the alkynyl group. The method was applicable to the synthesis of dibenzopentalene derivatives as well as various indenone derivatives. A mechanism for the reaction is discussed.

Biocatalytic asymmetric amination of carbonyl functional groups – a synthetic biology approach to organic chemistry

Transaminases catalyze amino transfer reactions from amino donors such as amino acids or amines to keto acids or ketones to give chiral amino acid or amines in optically pure form. alpha-Amino acid dehydrogenases catalyze the asymmetric reductive amination of alpha-keto acids using ammonia as amino donor to furnish L-amino acids. The distinct features and synthetic application of these two enzymes are reviewed in an effort to illustrate their promising and challenging aspects in serving as approaches to the direct asymmetric synthesis of optically pure amines from the corresponding keto compounds, a formidable problem in organic chemistry.

Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter pylori

Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

Zinc‐Mediated Intramolecular Acyl and Amino Transfer Reactions of Aryl Iodides.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Methionine regeneration and aspartate aminotransferase in parasitic protozoa.

Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.

The Methionine Chain Elongation Pathway in the Biosynthesis of Glucosinolates in Eruca sativa (Brassicaceae)

Glucosinolates are nitrogen- and sulfur-containing plant natural products that have become increasingly important in human affairs as flavor precursors, cancer-prevention agents, and crop protectants. While many glucosinolates are biosynthesized from common amino acids, the major glucosinolates in economically important species of the Brassicaceae, such as Brassica napus (oilseed rape), are thought to be formed from chain-elongated derivatives of methionine or phenylalanine. We investigated the chain elongation pathway for methionine that is involved in glucosinolate biosynthesis in Eruca sativa. Isotopically labeled methionine and acetate were administered to cut leaves and the major product, 4-methylthiobutylglucosinolate (isolated as its desulfated derivative), was analyzed by MS and NMR. Administration of [U-13C]methionine showed that the entire carbon skeleton of this amino acid, with the exception of the COOH carbon, is incorporated as a unit into 4MTB. Administration of [13C]- and [14C]acetate gave a labeling pattern consistent with the operation of a three-step chain elongation cycle which begins with the condensation of acetyl-CoA with a 2-oxo acid derived from methionine and ends with an oxidative decarboxylation forming a new 2-oxo acid with one additional methylene group. Administration of [15N]methionine provided evidence for the transfer of an amino group to the chain-elongated 2-oxo acid, forming an extended amino acid which serves as a substrate for the remaining steps of glucosinolate biosynthesis. The retention of a high level of 15N in the products suggests that the amino transfer reactions and the chain elongation cycle are localized in the same subcellular compartment.

Cloning two genes for nicotianamine aminotransferase, a critical enzyme in iron acquisition (Strategy II) in graminaceous plants.

Nicotianamine aminotransferase (NAAT), the key enzyme involved in the biosynthesis of mugineic acid family phytosiderophores (MAs), catalyzes the amino transfer of nicotianamine (NA). MAs are found only in graminaceous plants, although NA has been detected in every plant so far investigated. Therefore, this amino transfer reaction is the first step in the unique biosynthesis of MAs that has evolved in graminaceous plants. NAAT activity is dramatically induced by Fe deficiency and suppressed by Fe resupply. Based on the protein sequence of NAAT purified from Fe-deficient barley (Hordeum vulgare) roots, two distinct cDNA clones encoding NAAT, naat-A and naat-B, were identified. Their deduced amino acid sequences were homologous to several aminotransferases, and shared consensus sequences for the pyridoxal phosphate-binding site lysine residue and its surrounding residues. The expression of both naat-A and naat-B is increased in Fe-deficient barley roots, while naat-B has a low level of constitutive expression in Fe-sufficient barley roots. No detectable mRNA from either naat-A or naat-B was present in the leaves of either Fe-deficient or Fe-sufficient barley. One genomic clone with a tandem array of naat-B and naat-A in this order was identified. naat-B and naat-A each have six introns at the same locations. The isolation of NAAT genes will pave the way to understanding the mechanism of the response to Fe in graminaceous plants, and may lead to the development of cultivars tolerant to Fe deficiency that can grow in calcareous soils.

Mechanistic features of pepsin-catalyzed amino transfer reactions.

Peptic cleavage of N-trifluoroacetyl-L-tryptophan (CF3CO-Trp) at pH 2.45 to 5.4 in the presence of L-beta-phenyllactic acid (Pla) yields much PlaTrp. Formation of PlaTrp represents a typical peptic amino transfer reaction. In this instance PlaTrp undoubtedly derives from a reaction between the acceptor, Pla, and the carboxylate anion, CF3CO-TrpCOOO- (or a species obtained from it). This observation suggests that in general the anionic form of a substrate, such as AcPheTrpCOOO- in the case of AcPheTrp, may be the primary source of the amino acid residue transferred in amino transpeptidations. Other evidence supports the validity of the proposal. Furthermore it has the virtue of rationally explaining why AcPheTrpNH2-like substrates fail to participate in amino transpeptidations and why these reactions tend to increase in importance at high pH.

Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products.

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.

Functional properties of ornithine-ketoacid aminotransferase from rat liver

Functional properties of rat liver ornithine ketoacid aminotransferase ( l-ornithine:2-oxoacid aminotransferase, EC 2.6.1.13) were examined by two procedures: (A) The overall enzyme reaction was separated into its component half-reactions and the capacity of the enzyme to react with each of its substrates individually was investigated under different conditions. (B) The enzyme was exposed to pyridoxal phosphate, and resultant effects on enzyme activity and pyridoxal phosphate binding were analyzed. The following properties of the enzyme were disclosed by these studies. 1. 1. The reaction of the enzyme with ornithine alone is maximal between pH 8 and 10 whereas its reaction with α-ketoglutarate is maximal between pH 6 and 8. The pH optimum for the overall reaction (pH 8.15) coincides with the point of intersection of the curves for the half reactions. 2. 2. The pyridoxamine form of the enzyme, produced by the reaction of the pyridoxal enzyme with ornithine, reverts to the pyridoxal form in the absence of the second substrate. The rate of this reversion is lower in Tris than in phosphate buffer, although the catalytic activity of the enzyme is identical in both buffers. The lability of pyridoxamine at the active site is a consequence of its attachment to the apoenzyme, since free pyridoxamine phosphate does not release its amino group under the same conditions. It is suggested that this destabilizing effect of the apoenzyme is important to its role in the catalysis of the amino-transfer reaction. 3. 3. Exposure of the enzyme to excess pyridoxal phosphate is accompanied by the partial inactivation of the enzyme. This partial inactivation coincides with the binding of a small amount of pyridoxal phosphate to the enzyme. The further binding of pyridoxal phosphate produces no additional inactivation. Since pyridoxal phosphate reacts specifically with lysine residues in proteins the results suggest that certain of the lysine residues are involved in maintaining the enzyme at full catalytic capacity while others are functionally inert.

Glyoxylate aminotansferases from oat leaves.

Alanine : glyoxylate, glycine : 2-oxoglutarate (EC 2.6.1.4), and serine : glyoxylate aminotransferases were isolated and partially purified by two different procedures from oat leaves (Avena sativa L. var. Garry). Initial velocity, enzyme resolution and reconstitution, pyridoxal 5-phosphate activation, and enzyme inhibitor studies showed that the serine : glyoxylate aminotransfer reaction operates by the Ping Pong Bi Bi mechanism and involves the coenzyme pyridoxal phosphate, as do other aminotransferases, but does not appear to involve metal ions or sulfhydryl groups. Enzyme specificity determinations suggested that glyoxylate aminotransfer reactions are catalyzed by protein(s) distinct from other aminotransferases. Results are discussed in relation to earlier work with comparable enzymes from wheat leaves and to similar studies with enzymes from animal sources.