Structural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass.

Oliver W Sterritt,Timothy M Ryan,Eric J.M Lang,Emily J Parker,Geoffrey B Jameson,Borries Demeler,Sarah A Kessans

doi:10.1042/bsr20181605

Abstract

In Pseudomonas aeruginosa (Pae), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α2a and α2b, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.

Highlights

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen often associated with the chronic infection of patients suffering from cystic fibrosis [1]
A second distinct group of sequences, which are distant from the main cluster, is evident. Sequences from this second grouping are shorter in sequence length, relative to those found in the main type II D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) cluster, due to the predicted omission of the sequence corresponding to the α2a and α2b helices
The structure of PaeDAH7PSPA1901 further highlights the complex evolutionary trajectory for the type II DAH7PSs that has delivered type II enzymes which exhibit a diverse range of quaternary assemblies, and associated allosteric functionalities, required to support the efficient production of chorismate within either primary or secondary metabolism

Summary

Introduction

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen often associated with the chronic infection of patients suffering from cystic fibrosis [1]. The extra-barrel elements in MtuDAH7PS provide three distinct allosteric binding sites, on the single enzyme, that are each selective for either Trp, Tyr or Phe, and together they contribute towards a complex allosteric regulatory mechanism where binary or ternary combinations of aromatic amino acids that include Trp act synergistically to inhibit the enzyme [34,35,36] These extensions are responsible for the formation of the oligomeric interfaces that are present in the homotetrameric assemblies of the characterised type II enzymes. These DAH7PSs appear to contain an N-terminal extension to the core (β/α)8-barrel, comparable with that observed in PaeDAH7PSPA2843, but lack sequence corresponding to the inserted helices α2a and α2b for PaeDAH7PSPA2843 This difference in accessory structural elements may have consequences for the formation of the quaternary assembly and for allosteric functionality. The distinct structural and functional properties of PaeDAH7PSPA1901, in combination with sequence characteristics of the type II DAH7PSs, suggest that the type II DAH7PSs be further classified into two groups noted here as type IIA and type IIB

Experimental procedures

Results and discussion

Conclusion