Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue

Abstract

Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including b-alanine:pyruvate and g-aminobutyrate:a-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP·PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a wide range of chemical reactions, including the racemisation and decarboxylation of amino acids and transamination between amino acid donors and keto acid acceptors, [1,2] and new reactions, including oxidations, [3] continue to be discovered. Some of these enzymes, notably those of the "PLP fold type I" and belonging to the class III transaminase (TA) subgroup, have become extremely useful in biotechnology, because some members possess the ability to form chiral amines from ketone precursors, [4] whereas others catalyse the useful racemisation of amino acid amides. [5,6] In a review in 2015, Steffen-Munsberg and co-workers also drew attention to more obscure and uncharacterised reactions of the class III transaminase group, [1] including roles as phospholyases (E.C. 4.2.3.2). Phospholyases had been identified and partially characterised in early work by Jones [7] and Faulkner [8] in strains of Erwinia and Pseudomonas.The Erwinia enzyme was reported to catalyse the transformation of phosphoethanolamine (PEtN, 1) to yield acetaldehyde (2), ammonia and phosphate (Scheme 1).The involvement of PLP in the elimination of phosphate from 1 has since been established for vertebrate enzymes such as human AGXT2L1 and AGXT2L2 by the groups of Schaftingen [9] and Peracchi.[10] These enzymes have a role in phospholipid metabolism, and are of interest as playing a role in neuropsychiatric disorders, [9] although no structure of such a PLPdependent phospholyase has yet been reported. As part of an ongoing study of the structural and catalytic diversity displayed by PLP-dependent enzymes, we cloned the complement of genes encoding predicted transaminase enzymes from the bacterium Arthrobacter aurescens TC1 [11] into Escherichia coli, and many of the genes were expressed in the soluble fraction. From detailed bioinformatics analysis of this enzyme complement by previously described methods, [1] and from direct comparison with the AGXT2L1 sequence, [9,10] it was predicted that th...

The Arabidopsis thaliana gene annotated by the locus tag At3g08860 encodes alanine aminotransferase

“…ClustalW alignment of top five best hits was used to create the images with BOXSHADE (https://embnet.vital-it.ch/software/BOX_form.html). Three‐dimensional homology model for the At3g08860 protein was constructed via SWISS‐MODEL (Schwede, Kopp, Guex, & Peitsch, ) after alignment to the Arthrobacter aurescens TC1 phospholyase template, a class III transaminase homolog (PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5G4I) (Cuetos et al, ). The template was chosen as the crystal structure with the best sequence identity (35.82%) to the enzyme based on a search with HHBlits against the SWISS‐MODEL template library (Remmert, Biegert, Hauser, & Söding, ).…”

Structure and Kinetics of the S-(+)-1-Amino-2-propanol Dehydrogenase from the RMM Microcompartment of Mycobacterium smegmatis

“…While no genomic sequence exists for the strain investigated in their study, our analysis indicates that RMM microcompartments are absent from all known γ-Proteobacterial genomes, while γ-Proteobacteria, including Pseudomonas strains, do routinely contain APDH homologues outside of RMM operons (cluster I in Figure C) that associate with aminotransferase and phosphotransferase homologues. Phosphoethanolamine phospholyase was recently structurally characterized, and the key determinant of the phospholyase activity was found to be the presence of a basic pocket that accommodates the phosphate group of the substrate . The M. smegmatis aminotransferase from the non-RMM gene cluster (MSM0782) is 42% identical to this enzyme, with all of the catalytic residues and basic phosphate-binding residues conserved; this protein is then a candidate aminopropanol O-phosphate phospholyase.…”

“…Phosphoethanolamine phospholyase was recently structurally characterized, and the key determinant of the phospholyase activity was found to be the presence of a basic pocket that accommodates the phosphate group of the substrate. 29 The M. smegmatis aminotransferase from the non-RMM gene cluster (MSM0782) is 42% identical to this enzyme, with all of the catalytic residues and basic phosphate-binding residues conserved; this protein is then a candidate aminopropanol Ophosphate phospholyase. The phosphotransferase protein also found in this gene cluster lacks any well-characterized close homologues, but a role as a aminopropanol kinase is not unreasonable given that other family members typically phosphorylate aminoglycosides.…”

“…Comparing non-RMM-associated clusters with the RMM-associated counterparts in M. smegmatis, the permease proteins share about 64% identity, the phosphotransferases share approximately 45% identity, and the aminotransferases share 27% identity. The RMM-associated aminotransferases in particular do not conserve most of the basic residues found in the ethanolamine phospholyase catalytic pocket, 29 meaning, if they are phospholyases, they have evolved convergently and may exhibit substantive mechanistic differences. An aminopropanol kinase encapsulated in a microcompartment would also require access to ATP: microcompartments typically recycle cofactors such as NADP(H) and CoA, 11 but there are no candidate enzymes known to be associated with the RMM that might regenerate ATP.…”

Structural and kinetic characterization of (S)-1-amino-2-propanol kinase from the aminoacetone utilization microcompartment of Mycobacterium smegmatis

“…The only well characterized example is ethanolamine-phosphate phospho-lyase (EC 4.2.3.2.) (25,26), a homolog of class III aminotransferases. Strongly supporting this idea, propanolamine phosphate phospho-lyase activity was found associated with APDH, APK, and propionaldehyde dehydrogenase activities in enriched Pseudomonas or Erwinia extracts by Turner and colleagues (17,18) in the 70's.…”