Discovery of a Ni2+-dependent guanidine hydrolase in bacteria

Abstract

Nitrogen availability is a growth-limiting factor in many habitats 1 , and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogenrich compound guanidine occurs widely in nature 2-4 , but its utilization is impeded by pronounced resonance stabilization 5 , and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni 2+ -dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni 2+ instead of the typical Mn 2+ ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats.

Guanidino acid hydrolysis by the human enzyme annotated as agmatinase

“…However, kinetic constants in this range do not allow to draw conclusions about a potential physiological relevance. For example, we recently reported that despite its relatively high K M the guanidine hydrolase GdmH allowed Synechocystis to utilize guanidine as sole nitrogen source 8 . The analysis of further substrates that are not known to occur in humans indicated that the activity of GDAH strictly depends on a negatively charged functionality opposite to the guanidine group.…”

“…It has been noted before that AGMAT activity has never been demonstrated conclusively and it was speculated by others that so-far unidentified co-factors might be necessary for its activity 7 . For the recently discovered guanidine hydrolase, also a member of the ureohydrolase family, we showed that its activity depends on the chaperone-mediated insertion of Ni 2+ rather than Mn 2+ into the active site 8 . It is conceivable that isolated or recombinant AGMAT similarly lacks essential factors for its activity.…”

Still rocking in the structural era: A molecular overview of the small multidrug resistance (SMR) transporter family

“…But the molecular players have only begun to emerge since 2017, beginning with the discovery of riboswitch-controlled operons dedicated to Gdm + metabolism and transport ( 45 ). Four unrelated classes of Gdm + riboswitches have been identified ( 45 , 47 , 48 , 49 , 50 ), along with three distinct enzymatic pathways for utilizing Gdm + as a nitrogen source ( 51 , 52 , 53 , 54 ), including as a sole nitrogen source by some bacteria ( 53 , 55 ). The bacteria that do not consume Gdm + —about half of those with Gdm + riboswitches—instead produce and export endogenous Gdm + , likely as a metabolic waste product ( 5 , 45 ).…”

“…The riboswitches bind Gdm + with K D values between ∼60 to 200 μM ( 45 , 47 , 48 , 49 ) to upregulate expression of the associated transporters and enzymes. These proteins have somewhat higher K m values for Gdm + , between ∼200 μM −1 mM ( 4 , 45 , 51 , 53 ), suggesting that Gdm + accumulation becomes toxic to cells and must be mitigated within this range.…”

Canavanine utilization via homoserine and hydroxyguanidine by a PLP-dependent γ-lyase in Pseudomonadaceae and Rhizobiales

“…[17][18][19][20][21] Moreover, selective degradation pathways exist to utilize guanidine as nitrogen sources. 17,[22][23][24] Following the clarification of guanidine degradation, we are currently investigating biotic sources of guanidine and close derivatives. The present work describes the first specific canavanine degradation pathway.…”