# Previously Uncharacterised Aliphatic Amino Acid Positions Modulate the Apparent Catalytic Activity of the EAL Domain of ZMO_1055 and Other Cyclic Di‐GMP‐Specific EAL Phosphodiesterases

## TL;DR

This paper shows that specific amino acid changes in the EAL domain of bacterial proteins can reverse their catalytic activity, affecting cyclic di-GMP signaling.

## Contribution

The study identifies previously uncharacterized aliphatic amino acid positions that modulate EAL domain activity in cyclic di-GMP signaling.

## Key findings

- The A526V substitution reduces the catalytic activity of the ZMO1055 EAL domain.
- Substituting A526 with amino acids with longer aliphatic side chains reverts to diguanylate cyclase activity.
- These substitutions affect bacterial traits like flocculation, biofilm, and motility.

## Abstract

The nearly ubiquitous second messenger cyclic di‐GMP signalling system decides about the bacterial lifestyle transition between sessility and motility. GGDEF diguanylate cyclase and EAL phosphodiesterase domains conventionally conduct the turnover of the signalling molecule being subject to micro‐ and macroevolution. While highly conserved signature amino acids involved in divalent cation binding and catalysis have readily been identified, recognition of single amino acid substitutions that modulate the catalytic activity has been rare. Associated with development towards cellulose‐mediated self‐flocculation in
Zymomonas mobilis
ZM401, the A526V substitution and substitutions with amino acids with longer aliphatic side chains gradually revert the apparent catalytic activity of the EAL domain in the PAS‐GGDEF‐EAL ZMO1055 phosphodiesterase as monitored by flocculation, biofilm and motility assays. Remarkably, the effect of the A526V substitution equivalent is observed among other investigated GGDEF‐EAL proteins. Furthermore, substitutions of aliphatic side chain amino acids at distinct alternative positions affect ZOM1055 activity, while the M525L substitution has a context‐dependent effect. Thus, single amino acid substitutions outside of signature amino acid positions can even revert the target output and thus significantly contribute to the flexibility and adaptability of the cyclic di‐GMP signalling network. At a phylogenetic scale, ZMO1055 homologues seem to be a current evolutionary target.

The A526V substitution, previously not recognised to affect the functionality of the EAL domain, downregulates the apparent catalytic activity of the PAS‐GGDEF‐EAL ZMO1055 phosphodiesterase and other GGDEF‐EAL proteins. Substitution of A526, which is conserved among homologues, with amino acids with longer aliphatic side chains than valine, reverts to an apparent diguanylate cyclase activity.