Fig 1.
The PLP-dependent generation of the enamine 2 aminoacrylate from serine. Enamine/imine intermediates (2-aminoacrylate/iminopropionate) are in equilibrium and the latter is hydrolyzed by solvent water or facilitated by RidA protein, resulting in production of pyruvate. 2AA accumulation in an S. enterica ridA mutant is responsible for the inactivation of particular PLP-enzymes, which leads to growth defects.
Table 1.
Strains and plasmids.
Table 2.
Primers used in plasmid construction.
Fig 2.
Sequence alignment of Rid proteins.
Amino acid sequences of the Rid proteins used in this study are aligned and grouped by subfamily (RidA or Rid1-3). Similar residues are boxed in grey and identical residues are boxed in black. Active site residues proposed for S. enterica RidA are marked with asterisks. The amino acid sequences were aligned using Clustal Omega [39] and residues were shaded using the ExPASy BoxShade server.
Table 3.
Rid subfamily representatives and identity to S. enterica RidA.
Fig 3.
Rid proteins deaminate 2AA in vitro.
Reaction mixtures (100 μL) contained Tris-HCl (100 mM, pH 8), NADH (250 μM), pyridoxal 5’-phosphate (30 μM), pyruvate kinase/lactate dehydrogenase (5 Units) and purified cysteine desulfhydrase CdsH (0.27 μM). Purified Rid protein (0.19 μM) was added as indicated. (A) Absorbance was monitored at 340 nm for 60 seconds following the addition of L-cysteine to 1 mM final concentration. Reactions contained CdsH and purified Rid proteins as indicated. (B) The initial rate of pyruvate formation for each reaction was calculated using the molar extinction coefficient (ε = 6,200 M-1 cm-1) for NADH oxidation during the first 30 seconds. The mean of three replicates is plotted and error bars indicate standard deviation.
Fig 4.
Representative RidA and Rid1 proteins rescue growth of S. enterica ridA mutant strain.
An S. enterica ridA strain (DM12920) was transformed with pBAD24 constructs harboring no insert (pBAD24), S. enterica ridA, P. aeruginosa PA0814, A. baylyi ACIAD3089, P. syringae PSPTO_0102, P. aeruginosa PA5083, P. fluorescens PFL_1385, or P. syringae PSPTO_3006. The strains were grown in minimal glycerol medium supplemented with (A-D) 5 mM serine or (E-H) 250 μM cysteine, with (closed symbols) or without (open symbols) arabinose. The corresponding Rid subfamily assignment is presented above each graph. Growth was monitored by optical density at 650 nm with shaking at 37°C. Error bars indicate standard deviation for three biological replicates.
Fig 5.
Reaction schemes for FAD-dependent oxidase.
The reaction mechanism shows the FAD-dependent production of an imine from an amino acid. Imines are hydrolyzed by solvent water or facilitated by Rid proteins, or in the presence of semicarbazide react to form a semicarbazone compound. R’ represents the amino acid functional group.
Table 4.
Rid proteins deaminate a range of imine substratesa.
Fig 6.
Rid2 and Rid3 proteins deaminate iminoarginine produced by DauA.
Assay mixtures (100 μL) were monitored in microtiter plate format and contained potassium pyrophosphate (50 mM, pH 8.7), neutralized semicarbazide (10 mM), bovine liver catalase (24 Units) and ~1 μM DauA (1.7 μM total protein) from the partial purification. (A) Rid protein (10 μM) was added and the substrate was D-arginine (1 mM). Following the addition of substrate, the path length of each well was measured and the change in absorbance at 248 nm was monitored for ten minutes. (B) The bar graph shows the rate of semicarbazone formation calculated from the observed rate of product formation at 248 nm using the molar extinction coefficient (ε = 10,300 M-1 cm-1). (C) The rate of semicarbazone formation is presented as a percentage of the rate observed in the control (DauA alone) reaction mixture that lacks Rid proteins. All reaction mixtures contained ~ 1 μM DauA and were initiated with 1 mM D-arginine. The effect of purified Rid2 and Rid3 protein concentration on the rate is shown. Error bars indicate standard deviation of three replicates and curves were fitted using the equation for one phase exponential decay using Prism.
Fig 7.
Working model for Rid2 and Rid3 activity.
The FAD-dependent D-arginine dehydrogenase generates iminoarginine, which is deaminated by solvent water or by Rid proteins (Rid2 or Rid3), resulting in 2-ketoarginine formation. In Pseudomonas aeruginosa, DauB converts 2-ketoarginine to L-arginine, enabling the organism to grow on D-arginine as the sole carbon and nitrogen source [44].