6-hydroxy-L-homoarginine(1+)

Expression of plant EFE-like proteins causes guanidine production in E. coli

The detection of free guanidine indicated that 2-ODD-C23 might be homoarginine-6-hydroxylases, as 6-hydroxyhomoarginine is expected to decay spontaneously to yield guanidine and AASA. The latter is in spontaneous equilibrium with P6C, which can be converted to pipecolate by P5CR (proC in E. coli, ). Indeed, we detected pipecolate in the E. coli cultures in quantities proportional to the amount of guanidine (). Only in the cells expressing Din11s, the proportion of pipecolate was lower, suggesting that Din11s used both arginine and homoarginine as substrates. In contrast to guanidine, pipecolate was predominantly retained inside the cells (). To analyze if the plant enzymes can convert 2-oxoglutarate to ethylene, we compared bacterial cultures expressing either PsEFE or one of the Arabidopsis enzymes (). By GC-FID analysis of the headspace of bacterial cultures expressing EFE, Din11s, At3g49630, or At3g50210 and incubated with 1 mM arginine or homoarginine for 18 hr in air-tight tubes, we observed the formation of up to 0.18% (v/v) ethylene by cultures expressing EFE. In contrast, none of the plant enzymes produced even traces of ethylene [detection limit 0.0002% (v/v)]. As expected, the cultures expressing EFE or Din11s contained guanidine, irrespective of the supplement. The cultures expressing At3g49630 or At3g50210 only contained guanidine, when they were fed with homoarginine.

Introduction

Only two enzymes are known to release guanidine from arginine in side reactions. The Ethylene Forming Enzyme (EFE) from Pseudomonas savastanoi converts 2-oxoglutarate (2-OG) into ethylene and this activity depends on structural changes induced by binding of arginine to the active site (; ). In a second reaction with roughly 50% turnover rate, EFE also catalyzes the hydroxylation of arginine at the C5 atom, yielding unstable 5-hydroxyarginine, which spontaneously decays to glutamate-5-semialdehyde (GSA) and guanidine. The arginine-4,5-desaturase NapI predominantly catalyzes desaturation of the C4-C5 bond but also produces a small proportion unstable 5-hydroxyarginine (). Both enzymes belong to the class of Fe2+ and 2-OG-dependent dioxygenases (2-ODD), which use molecular oxygen to catalyze a broad range of reactions typically involving an FeIV-oxo-intermediate formed after the initial conversion of 2-OG to succinate (; ; ). Other enzymes from this family, like VioC or OrfP, catalyze the hydroxylation of arginine at C3 or C3 and C4, respectively, without initiating guanidine release (; ). A third guanidine-producing enzyme, the guanylurea hydrolase GuuH from Pseudomonas mendocina strain GU, is from a different protein family and may have evolved recently to utilize guanylurea generated by metformin degradation ().