Steady-State Kinetic Mechanism and Reductive Half-Reaction of d-Arginine Dehydrogenase from Pseudomonas aeruginosa

Abstract

D-arginine dehydrogenase from Pseudomonas aeruginosa catalyzes the oxidation of D-arginine to iminoarginine, which is hydrolyzed in solution to ketoarginine and ammonia. In the present study, we have genetically engineered an untagged form of the enzyme that was purified to high levels and characterized in its kinetic properties. The enzyme is a true dehydrogenase that does not react with molecular oxygen. Steady-state kinetic studies with D-arginine or D-histidine as substrate and PMS as the electron acceptor established a ping-pong bi-bi kinetic mechanism. With the fast substrate D-arginine a dead-end complex of the reduced enzyme and the substrate occurs at high concentrations of D-arginine yielding substrate inhibition, while the overall turnover is partially limited by the release of the iminoarginine product. With the slow substrate D-histidine the initial Michaelis complex undergoes an isomerization involving multiple conformations that are not all equally catalytically competent for the subsequent oxidation reaction, while the overall turnover is at least partially limited by flavin reduction. The kinetic data are interpreted in view of the high-resolution crystal structures of the iminoarginine--and iminohistidine--enzyme complexes.

Importance of glutamate 87 and the substrate α-amine for the reaction catalyzed by d-arginine dehydrogenase

“…D-Leucine d10 was obtained from CDN Isotopes (Pointe-Claire, Canada). PaDADH-wt was prepared as described previously [2,6]. All other reagents used were of the highest purity commercially available.…”

“…Flavin reduction is irreversible with D-histidine or D-leucine as substrate, as established by a zero intercept value in a plot of the observed rate constant for flavin reduction as a function of substrate concentration [6]. With D-arginine, however, the reaction is too fast and cannot be studied in a stopped-flow spectrophotometer due to >80% of the flavin being completely reduced within the mixing time of the instrument (i.e., 2.2 ms), as previously reported [6]. Amine oxidation has been http://dx.doi.org/10.1016/j.abb.2015.01.017 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.…”

“…L-Amino acids are not substrates for PaDADH [2]. PaDADH is a strict dehydrogenase because during turnover it reacts with an electron acceptor other than molecular oxygen, presumably ubiquinone in vivo [6]. The enzyme follows a Ping-Pong Bi-Bi steadystate kinetic mechanism, as demonstrated with D-arginine or D-histidine as substrate and phenazine methosulfate (PMS) as electron acceptor [6].…”

“…PaDADH is a strict dehydrogenase because during turnover it reacts with an electron acceptor other than molecular oxygen, presumably ubiquinone in vivo [6]. The enzyme follows a Ping-Pong Bi-Bi steadystate kinetic mechanism, as demonstrated with D-arginine or D-histidine as substrate and phenazine methosulfate (PMS) as electron acceptor [6]. In the reductive half-reaction the enzyme-bound flavin (PaDADH ox ) is reduced to hydroquinone with concomitant oxidation of the amino acid to the imino acid (PaDADH ox -P), as shown in Scheme 2.…”

Utilization of d -Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans

“…To confirm the classification of GOX1253 and GOX2071, their abilities to use O 2 as a direct electron acceptor were assessed using a Clark-type oxygen electrode as described in a previous study (34). Purified enzyme (0.016 U/ml) was added to a reaction chamber containing 50 mM Tris-HCl (pH 7.4) and 20 mM D-lactate.…”