Type I pyridoxal 5'-phosphate dependent enzymatic domains embedded within multimodular nonribosomal peptide synthetase and polyketide synthase assembly lines

Abstract

Background: Pyridoxal 5'-phosphate (PLP)-dependent enzymes of fold type I, the most studied structural class of the PLP-dependent enzyme superfamily, are known to exist as stand-alone homodimers or homotetramers. These enzymes have been found also embedded in multimodular and multidomain assembly lines involved in the biosynthesis of polyketides (PKS) and nonribosomal peptides (NRPS). The aim of this work is to provide a proteome-wide view of the distribution and characteristics of type I domains covalently integrated in these assemblies in prokaryotes.

Results: An ad-hoc Hidden Markov profile was calculated using a sequence alignment derived from a multiple structural superposition of distantly related PLP-enzymes of fold type I. The profile was utilized to scan the sequence databank and to collect the proteins containing at least one type I domain linked to a component of an assembly line in bacterial genomes. The domains adjacent to a carrier protein were further investigated. Phylogenetic analysis suggested the presence of four PLP-dependent families: Aminotran_3, Beta_elim_lyase and Pyridoxal_deC, occurring mainly within mixed NRPS/PKS clusters, and Aminotran_1_2 found mainly in PKS clusters. Sequence similarity to the reference PLP enzymes with solved structures ranged from 24 to 42% identity. Homology models were built for each representative type I domain and molecular docking simulations with putative substrates were carried out. Prediction of the protein-protein interaction sites evidenced that the surface regions of the type I domains embedded within multienzyme assemblies were different from those of the self-standing enzymes; these structural features appear to be required for productive interactions with the adjacent domains in a multidomain context.

Conclusions: This work provides a systematic view of the occurrence of type I domain within NRPS and PKS assembly lines and it predicts their structural characteristics using computational methods. Comparison with the corresponding stand-alone enzymes highlighted the common and different traits related to various aspects of their structure-function relationship. Therefore, the results of this work, on one hand contribute to the understanding of the functional and structural diversity of the PLP-dependent type I enzymes and, on the other, pave the way to further studies aimed at their applications in combinatorial biosynthesis.

Figure 1

Structurally Conserved Regions. Alignment of the Structurally Conserved Regions (SCR) of the 31 fold type I structures considered. Colors indicate conservation of residue physico-chemical properties. Each structure is labeled by its PDB code flanked by the sequence positions encompassing the reported SCRs. “SCR line” numbers the 13 conserved regions; below is the conservation histogram and the consensus sequence. The identically conserved residues in position 69 and 85 are the Asp interacting with the cofactor pyridine nitrogen and the Lys forming the Schiff base, respectively. Indels are not shown for easing the interpretation of the figure. Indel positions are denoted by the all-gap columns separating the different SCRs.

Figure 2

Topology of the unrooted consensus tree calculated from the multiple alignment of the non-redundant set of type I domains. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches whenever the value was greater than 50. Sequences are labeled by their UniProt code and the following information, in order: specie name, phylum, specificity, cluster type and product (definitions refers to those reported in Table 2). Question mark denotes unknown information. Red circles indicate reference structures identified by their PDB id codes: [PDB:2E7U] is glutamate-1-semialdehyde 2,1-aminomutase from Thermus termophilus; 1DGE, dialkylglycine decarboxylase from Burkholderia cepacia; [PDB:1VEF], acetylornithine aminotransferase from Thermus termophilus; [PDB:3A2B], serine palmitoyltransferase from Sphyngobacterium multivorum; [PDB:1BS0], 8-amino-7-oxononanoate synthase from Escherichia coli; [PDB:3TQX], 2-amino-3-ketobutyrate coenzyme A ligase from Coxiella burnetii; [PDB:1C7G], tyrosine phenol-lyase from Erwinia herbicola; [PDB:2JIS], cysteine sulfinic acid decarboxylase from Homo sapiens. Subtrees defining the four families are drawn with different colours. The tree is unrooted.

Figure 3

Sequence alignment between a representative sequence of each family of type I domains and the most similar structural template. Sequences are labeled by their databank code. Aminotran_3 (a): [UniProt: Q2T5Z2] indicates polyketide synthase from Burkholderia thailandensis; [PDB:2E7U] is the glutamate-1-semialdehyde 2,1-aminomutase from Thermus thermophilus HB8. Aminotran_1_2 (b): [UniProt:B1XHP8] indicates AMP-binding enzyme from Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6); [PDB:3A2B] denotes serine palmitoyltransferase from Sphingobacterium multivorum. Beta_elim_lyase (c): [UniProt:F8TUA6] corresponds to keto-hydroxyglutarate-aldolase/polyketide synthase from Lysobacter sp.; [PDB:1C7G] labels the tyrosine phenol-lyase from Erwinia herbicola. Pyridoxal_deC (d): [UniProt:B6IZA3] is the non-ribosomal peptide synthetase module from Coxiella burnetii; [PDB:2JIS] stands for the cysteine sulfinic acid decarboxylase from Homo sapiens. Secondary structures are charted below the template sequence. Helices (alpha and 3₁₀ helices are designated by α or η respectively) are displayed as squiggles and beta strands (β) are rendered as arrows. Beta turns are denoted as “TT” and strict α turns as “TTT”. Dots indicate gaps. Identically conserved residues are displayed on a red background; red letters indicate conservative substitutions. Triangles mark residues known to be functionally important in the template enzyme. Black circles tag important residues from the other subunit. Stars label the Asp and the Lys residue involved in interaction with pyridine nitrogen and in Schiff-base forming, respectively. The black square in the panel (c) indicates the Arg381 of the template missing in the homologous PLP domain.

Figure 4

Structural superposition of the model-template pairs. Structural superposition of the model-template pairs for families Aminotran_3 (a), Aminotran_1_2 (b), Beta_elim_lyase (c) and Pyridoxal_deC (d) reported in Figure 3. Ribbon representation is used. Structural templates are colored in grey. Green and cyan indicate the model subunits. Cofactor is represented by transparent yellow spheres. Arrows point to insertion or deletion regions that are distinguished by magenta or yellow colors. The conformation of inserted magenta loop in the model of Aminotran_3 group (a) has no structural meaning: it has been modeled only with the purpose to indicate its approximate location on the protein surface.

Figure 5

Comparisons of the models of the active site of the domains representative of each type I subfamily. Grey drawing indicates the reference structural template, while orange and cyan depict the two subunits of the models. Cofactor is drawn as transparent yellow spheres encapsulating stick models. Relevant side chains are rendered as sticks. Numbering refers to Figure 3. (a) Type I domain from polyketide synthase from Burkholderia thailandensis [UniProt:Q2T5Z2], and glutamate-1-semialdehyde 2,1-aminomutase from Thermus thermophilus HB8 (internal aldimine) [PDB:2E7U]. (b) AMP-binding enzyme [UniProt:B1XHP8] from Synechococcus sp. (strain ATCC 27264/PCC 7002/PR-6), and serine palmitoyltransferase from Sphingobacterium multivorum (external aldimine with serine) [PDB-3A2B]. (c) keto-hydroxyglutarate-aldolase/polyketide synthase from Lysobacter sp. [UniProt:F8TUA6] and tyrosine phenol-lyase from Erwinia herbicola (internal aldimine) [PDB:1C7G]. (d) Nonribosomal peptide synthetase module from Coxiella burnetii [UniProt: B6IZA3] and cysteine sulfinic acid decarboxylase from Homo sapiens (internal aldimine) [PDB:2JIS].