Identification of Key Amino Acid Residues in L-Proline Catabolic Enzymes from Gram-positive Bacteria

Catabolism of the amino acid L-proline in most Gram-positive bacteria occurs through through a common pathway that involves the FAD-dependent activity L-proline dehydrogenase (PRODH) and the NAD+-dependent activity L-Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). The two activities are found in monofunctional proteins in the low G+C Firmicutes and in monofunctional or bifunctional proteins in the high G+C Actinobacteria. To assess the similarities of these proteins, the amino acid sequences from four representative species in the genera Bacillus, Halobacillus, Staphylococcus, Streptomyces, Arthrobacter/ Paenarthrobacter, Corynebacterium, and Mycobacterium were retrieved from the UniProtKB database and aligned. The resulting phylogenetic trees showed a good clustering of the four sequences for each of the seven genera. However, the 20 monofunctional proteins showed an overall sequence identity of only about 15% while the eight bifunctional proteins showed an identity of about 60%. To identify the key amino acid residues in these proteins, the sequences from the Gram-positive bacteria were aligned with the monofunctional proteins from Thermus thermophilus and Deinococcus radiodurans and the bifunctional or trifunctional proteins from Bradyrhizobium japonicum and Escherichia coli. These proteins from Gram-negative bacteria have been crystallized and used as models of catalytic activity. While there was very little overall sequence identity, the same key residues were found at both the PRODH and P5CDH active sites with only occasional conservative substitutions. Although the evidence for channeling the L-Δ1-pyrroline-5-carboxylate (P5C) product of the first reaction (PRODH) through a structural tunnel to the active site of the second reaction (P5CDH) is limited among the Gram-positive bacteria, sequence analysis indicated that key residues affecting this process are also present in the Gram-positive bacteria. This suggests that the basic mechanism for proline catabolism has been retained during evolution and is the same in almost all microorganisms.