Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation.

Ying Chen,Wei Hu,Kaikai Chi,Jin Peng,Jing He,Tiantian Su,Deyu Zhu,Shiheng Liu,Cuilan Liu,Yan Huang,Zhijie Xia,Lichuan Gu,Sujuan Xu

doi:10.1038/srep29499

Abstract

C-di-GMP (3’,5’ -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity.

Highlights

In addition to activation by oligomerization, the activity of GGDEF domains can be regulated by product inhibition
The PA2771 gene is located on the genomic island of strain PAO1, which has not been reported in strain PA14 and six other P. aeruginosa strains[22]
As expected, introducing a multi-copy plasmid carrying the PA2771 gene into the wild type strain (WT) and ΔPA2771 strains significantly decreased the swimming motility (Fig. 1a)

Summary

Introduction

In addition to activation by oligomerization, the activity of GGDEF domains can be regulated by product inhibition. The DGC activities of both PleD and WspR are regulated by non-competitive product inhibition via the binding of c-di-GMP to the allosteric I-site[20,21]. To study the mechanism of PA2771 catalysis and regulation, we crystallized full-length PA2771 and the GGDEF domain of PA2771 in complex with c-di-GMP.

Results

Conclusion