Inducible Degradation of Hydroxyproline in Pseudomonas putida: Pathway Regulation and Hydroxyproline Uptake

Abstract

Studies in Pseudomonas putida of the inducible degradation of hydroxyproline to a-ketoglutarate have indicated that either of the two epimers, hydroxy-L-proline or allohydroxy-D-proline, acts as an inducer of all the pathway enzymes. In a mutant lacking the first enzyme of the sequence, hydroxyproline-2-epimerase, which interconverts these two hydroxyproline epimers, either epimer is still equally active as an inducer of the remaining three enzymes, suggesting that each epimer has intrinsic inducer activity. The second and third enzymes of the sequence were induced coordinately. The induction process appeared to be insensitive to catabolite repression under a number of experimental conditions. The induced enzymes were stable even under conditions of nitrogen starvation and other conditions designed to increase protein turnover. In addition to inducing the degradative enzymes, the two hydroxyproline epimers were also found to induce an uptake system that concentrates hydroxyproline intracellularly. Either amino acid induced the uptake system for its epimer as well as for itself.

Properties of the Inducible Hydroxyproline Transport System of Pseudomonas putida

“…It was shown to lack activity for the first enzyme in the pathway for the degradation of hydroxy-L-proline. The characteristics of the mutant and the method used for its isolation were described in a previous paper (11). In addition, one experiment was conducted with another mutant, B-6, which has lost the ability to induce the degradative enzymes for hydroxy-L-proline (P. Koo and E. Adams, unpublished data).…”

“…L-Glutamic acid was purchased from Pfanstiehl Laboratories (Waukegan, Ill.), and hydroxy-L-proline A grade (free of proline) was purchased from Calbiochem (Los Angeles, Calif.). The preparation of 3H-allohydroxy-D-proline from hydroxy -L-proline -s -3H and the sources of other reagents were reported earlier (11). Hydroxy-L-proline-S-3H was obtained from New England Nuclear Corp. (Boston, Mass.).…”

“…Glutamate transport has been reported to be inducible in mycobacteria (21) and E. coli (12), and citrulline transport can be induced in Streptococcus faecalis (4). We have recently reported an inducible transport system for hydroxyproline in Pseudomonas putida (11).…”

“…The system can be induced in the mutant either by growth on allohydroxy-D-proline, the first degradative product, or by growth on an alternative carbon and nitrogen source in the presence of hydroxy-L-proline. These cells will then transport either epimer and will accumulate hydroxy-L-proline to an intracellular concentration 200 times that of the external medium (11).…”

Evidence for induced synthesis of an active transport factor for mandelate in Pseudomonas putida

“…Although specific transport systems for various substrates are known in Pseudomonas (Kogut & Podoski, 1953;Shilo & Stanier, 1957;Rosenfeld & Fiegelson, 1969;Gryder & Adams, 1969), an investigation of the uptake of mandelate by P. putida failed to demonstrate the existence of an active concentrating permease for this substance (Hegeman, 1966b). Radioactive mandelate entered both induced and uninduced cells very rapidly and the internal pool equilibrated with the external medium.…”