Molecular evolution of adenylating domain of aminoadipate reductase

Background Aminoadipate reductase (Lys2) is a fungal-specific protein. This enzyme contains an adenylating domain. A similar primary structure can be found in some bacterial antibiotic/peptide synthetases. In this study, we aimed to determine which bacterial adenylating domain is most closely related to Lys2. In addition, we analyzed the substitution rate of the adenylating domain-encoding region. Results Some bacterial proteins contain more than two similar sequences to that of the adenylating domain of Lys2. We compared 67 amino acid sequences from 37 bacterial and 10 fungal proteins. Phylogenetic trees revealed that the lys2 genes are monophyletic; on the other hand, bacterial antibiotic/peptide synthase genes were not found to be monophyletic. Comparative phylogenetic studies among closely related fungal lys2 genes showed that the rate of insertion/deletion in these genes was lower and the nucleotide substitution rate was higher than that in the internal transcribed spacer (ITS) regions. Conclusions The lys2 gene is one of the most useful tools for revealing the phylogenetic relationships among fungi, due to its low insertion/deletion rate and its high substitution rate. Lys2 is most closely related to certain bacterial antibiotic/peptide synthetases, but a common ancestor of Lys2 and these synthetases evolutionarily branched off in the distant past.


Background
Not only fungi, but also certain prokaryotes synthesize lysine through the 2-aminoadipate pathway [1][2][3]. However, the prokaryotic pathway is not identical to that of fungi. The fungal process required to synthesize lysine from 2-aminoadipate differs from that of prokaryotes [4]. The first step of this fungal-specific pathway is the reduction of 2-aminoadipate.
Aminoadipate reductase converts 2-aminoadipate to 2aminoadipate 6-semialdehyde via an adenosylated derivative. In Saccharomyces cerevisiae, this reaction requires Mg 2+ and the participation of the products of two genes, lys2 and lys5 [5]. Recently, it has been shown that aminoadipate reductase is encoded by only lys2, and that the Lys5 protein appears to be a specific phosphopantetheinyl transferase for Lys2, converting the inactive apo-Lys2 to the active holo-Lys2 [6,7].
The lys2 gene is a fungal-specific gene and generally appears to be present in a single copy in the genome. The Lys2 protein has no extensive homologous protein in eukaryotes, with the exception of fungi, but it does possess similarity to some bacterial antibiotic/peptide synthetases [4,[8][9][10]. Recently, Drosophila and mouse were found to have the analogue of Lys2, which function under degradation of lysine [11]. However, Lys2 is more similar bacterial antibiotic/peptide synthetases than the animal proteins. Lys2 has an adenylating, a peptidyl carrier, and a reductive domain. This protein has twelve conserved motifs. The adenylating domain contains nine conserved motifs [12]. In this study, we aimed to reveal which bacterial adenylating domain is the most closely related to Lys2.
In addition, in order to determine the substitution rate of lys2, we compared the lys2 sequences from closely related fungi. In this study, we sequenced lys2 fragments [13] and compared them among black-koji molds of the Aspergillus niger group. Comparisons between A. awamori and Penicillium chrysogenum (Table 1) and between A. awamori and A. fumigatus ( Table 2) showed that the rate of insertion/deletion in lys2 was lower and the nucleotide substitution rate was higher than that in ITS regions. We therefore believe that lys2 is a more powerful tool to reveal phylogenetic relationships among fungi than are the ITS regions.

Results and Discussion
The result of the homology search using BLAST showed that Lys2 had a more similar sequence to that of certain bacterial antibiotic/peptide synthetases than did any other existing proteins. In addition, some bacterial antibiotic/ peptide synthetases were shown to contain more than two homologous regions. For example, RS05859 in Ralstonia solanacearum GMI1000 has five homologous regions. Therefore, we obtained 57 amino acid sequences, with a value of E < 10 -25 , from 39 proteins (see Materials and Methods).  The phylogenetic tree (Fig. 1ab) shows that the adenylating domains from some bacterial antibiotic/peptide synthetases are distributed quite widely, and that duplications and/or horizontal transfers occurred many times. For example, Anabaena sp. PCC 7120 has 12 similar sequences within itself. In this tree, these 12 sequences were distributed among at least 6 groups. The present findings indicate that duplication and/or horizontal transfer occurred in the genome of Anabaena sp. PCC 7120. On the other hand, the adenylating domains from Lys2 formed a monophyletic cluster. However, the neighbor-joining tree presented here did not clarify which bacterial domain was most closely related to that of Lys2.
In order to determine which bacterial domain was most closely related to that of Lys2, a maximum likelihood analysis using PHYLIP version 3.6 [14] was carried out. We selected 27 amino acid sequences from the 67 sequences used in the neighbor-joining analysis. The alignment used in maximum likelihood analysis is shown in Fig. 2. The phylogenetic tree (Fig. 3) indicates that a protein (AGR L 2311) from Agrobacterium tumefaciens is most closely related to a common ancestor of Lys2, but this result had only weak bootstrap support (17%). In the bootstrap consensus tree (Fig. 3), the branch points at the early stage of evolution are very weak support. Animals and plants have no Lys2. If the common ancestor of eukaryotes had a similar protein, the other eukaryotes except for fungi had lost it.

Conclusions
This study indicated that Lys2 is more closely related to certain bacterial antibiotic/peptide synthetases than it is to any other known proteins. However, in the distant past, a common ancestor of Lys2 branched off from the bacterial antibiotic/peptide synthetase. This study did not find evidence for a direct horizontal transfer (i.e., at least not a recent horizontal transfer) between bacteria and a common ancestor of fungi. The lys2 gene has been inherited during fungal evolution. On the other hand, in the course of bacterial evolution, the duplication and/or horizontal transfer have occurred.

Materials and Methods
In

Figure 3
The bootstrap consensus tree among the selected 27 amino acid sequences based on the maximum likelihood analysis.