Evolutionary origin and functional diversification of aminotransferases

Abstract

Aminotransferases (ATs) are pyridoxal 5'-phosphate-dependent enzymes that catalyze the transamination reactions between amino acid donor and keto acid acceptor substrates. Modern AT enzymes constitute ∼2% of all classified enzymatic activities, play central roles in nitrogen metabolism, and generate multitude of primary and secondary metabolites. ATs likely diverged into four distinct AT classes before the appearance of the last universal common ancestor and further expanded to a large and diverse enzyme family. Although the AT family underwent an extensive functional specialization, many AT enzymes retained considerable substrate promiscuity and multifunctionality because of their inherent mechanistic, structural, and functional constraints. This review summarizes the evolutionary history, diverse metabolic roles, reaction mechanisms, and structure-function relationships of the AT family enzymes, with a special emphasis on their substrate promiscuity and multifunctionality. Comprehensive characterization of AT substrate specificity is still needed to reveal their true metabolic functions in interconnecting various branches of the nitrogen metabolic network in different organisms.

Keywords: PLP-dependent enzymes; amino acids; aminotransferases; enzyme evolution; nitrogen metabolism; transaminases.

Figure 1

Enzymatic and nonenzymatic transamination reactions, and the evolutionary history of the amino acid metabolism, proteinogenesis, and transamination.A, two half reactions of PLP-dependent transamination. In the first half reaction, an amino group from aspartate is transferred onto PLP, which generates oxaloacetate and PMP. In the second half, the amino group on PMP is transferred to pyruvate, forming alanine and regenerating PLP. B, evolution of transamination reactions since the origin of life. Transamination reactions were likely nonenzymatic initially and later catalyzed by hypothetical ribotransaminases during the RNA world, where the two-letter GC coded for a few amino acids. Additional amino acids were recruited after the genetic code expanded to three letters in the GCA phase during the RNA protein (RNP) world, when class I and II proto-ATs might have appeared and streamlined the amino acid metabolism. The subsequent GCAU phase and expansion of proteinogenic amino acids recruited class IV and class III ATs. About 4 billion years ago, LUCA inherited a diverse set of ATs and passed them down to its descendants. ATs underwent additional diversification during and after the Great Oxidation Event (GOE) and the appearance of eukaryotes and multicellularity. C, a nonenzymatic transamination reaction between aspartate and pyruvate, where aspartate is converted into an aldehydic acid (3-oxopropanoic acid), rather than keto acid (oxaloacetic acid). AT, aminotransferase; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate; RNP, ribonucleoprotein.

Figure 2

Metabolic roles of AT reactions in human, yeast, Arabidopsis, and E. coli. A metabolic map depicting AT enzyme functions in representative species. Proteinogenic amino acids are shown in gray ellipses. Thick solid and dashed arrows dictate the flow of nitrogen containing metabolites and catabolic pathways, respectively. ATs and pathways from E. coli, Arabidopsis, yeast, and human are shown with yellow, green, red, and blue, respectively. The AT enzyme abbreviations are listed in Table 1. AS, anthranilate synthase; AT, aminotransferase; DAPA, 7,8-diaminopelargonic acid; E4P, erythrose 4-phosphate; GDC, glutamate decarboxylase; GDH, glutamate dehydrogenase; GOGAT, glutamine oxoglutarate amidotransferase; GS, glutamine synthetase; 4HPP, 4-hydroxyphenylpyruvate; KAPA, 7-keto-8-aminopelargonic acid; α-KG, alpha-ketoglutarate; l,l-DAP, l,l-2,6-diaminopimelic acid; m-DAP, meso-2,6-diaminopimelic acid; MDH, malate dehydrogenase; 4MTOB, 4-methylthio-2-oxobutanoic acid; OA, oxaloacetate; PEP, phosphoenolpyruvate; 3PGA, 3-phosphoglyceric acid; R5P, ribose 5-phosphate; SSADH, succinic semialdehyde dehydrogenase; THDPA, tetrahydrodipicolinate; TS, tryptophan synthase; Ubq, ubiquinone.

Figure 3

Phylogeny and reported activities of ATs from representative organisms. Phylogenetic relationship of ATs from human (Hs), yeast (Sc), Arabidopsis (At), Escherichia coli (Ec), and Halobacterium volcanii (Hv). Multiple sequence alignment was performed using MAFFT-DASH (138), and the analysis was performed under default setting in MEGA 11 (343) neighbor-joining method (344, 345) with partial deletion site coverage set to 50% and 1000 bootstraps (346). Analysis was done separately for class III ATs from class I, II, and IV ATs because of their distinct evolutionary origins. ATs formed at least 12 distinct clades whose major substrates are shown. Activities detected in the literature for each enzyme are shown on the right. Red, orange, green, gray, and white denote major, side, predicted, absent, and untested activities, respectively. AT, aminotransferase.

Figure 4

Reaction mechanisms of AT-catalyzed transamination reaction. The first half-reaction mechanisms for class I, II, IV, and class III ATs are shown starting from the first amino acid substrate and PLP–enzyme complex (internal aldimine). After the formation of the Michaelis complex, the reaction proceeds through the formation of first and second geminal diamines and external aldimine. In class I, II, and IV ATs, the external aldimine first forms quinoid intermediate, which is subsequently converted to ketamine (blue arrows). In contrast, in class III ATs, external aldimine is directly converted to the ketimine through 1,3-prototropic shift mechanism (red arrows). Ketimine is subsequently converted to first and second carbinolamine intermediates and collapses to form PMP plus the first keto acid product. In the second half-reaction, PMP reacts with second keto acid substrate in the reverse direction to form second amino acid product and PLP. AT, aminotransferase; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate.

Figure 5

Structure and conserved residues of four classes of ATs.A, simplified secondary structures of AT polypeptides showing the overall topology of each class AT, which include α-helixes (boxes), β-sheets (arrows), and loops (straight lines). Black residues are conserved for >90% of aligned sequences within each class, except for the ones marked with a star, which are conserved for ∼70%. The Schiff-base lysine is shown in red and traced by redtraces. Functionally conserved glycine, glutamate/aspartate, and arginine are shown in orange and traced by orangetraces. Functional conservation of residues marked with Ϯ is inferred from the crystal structure. N- and C-terminal domains are shown in green and blue, respectively. B, overall structures of Escherichia coli class I aspartate AT (AAT; Protein Data Bank [PDB] ID: 1ARG (139)), class II GABA AT (GABT; PDB ID: 1SFF (347)), class IV phosphoserine AT (PSAT, PDB ID: 1BJO (141)), and class III BCAT (IlvE; 1i1l (142)). Non–active-site residues that are conserved within each AT class are labeled in red. C, conserved active-site residues (green) of the substrate (cyan)–PLP (dark blue) complex of four AT classes. The Schiff-base lysine is shown in red. IlvE is shown in complexes with both glutamate (right) and leucine (left). Note that in class I and IV, conserved arginine (R374 in AAT and R335 in PSAT) interacts with α-carboxylate of the substrate. The weakly conserved R in class II (R398 in GABT) does not interact with α-carboxylate of the substrate; instead another conserved arginine (R141 in GABT) fulfills this duty. In class III, a conserved tyrosine (Y96 in IlvE) interacts with α-carboxylate group of both glutamate and leucine, whereas the conserved arginine (R98 in IlvE) only interacts with the acidic side-chain carboxylate of glutamate. AT, aminotransferase; GABA, γ-aminobutyric acid; GABT, GABA AT; PLP, pyridoxal 5′-phosphate.

Figure 6

Sequence and structure features determining the substrate specificity of BCATs. Molecular modeling to predict binding modes of external aldimine intermediates of isoleucine (cyan) and phenylalanine (orange) with three homologous BCATs: A, Mycobacterium tuberculosis (PDB ID: 5U3F). B, Pseudomonas sp. UW4 (PDB ID: 6JIF). C, human mitochondria (PDB ID: 1KT8). D, putative amino acid residues of several BCATs consisting of the active sites deduced from sequence alignment. Color shading indicates physicochemical properties of amino acids: aliphatic/hydrophobic (pink), aromatic (yellow), glycine (orange), hydrophilic (green), and positive (blue). BCAT, branched-chain amino acid AT; PDB, Protein Data Bank.