Analyzing the Mechanistic Role of an Active Site Lysine Residue in Monofunctional and Bifunctional Proline Dehydrogenases

Abstract

The catabolism of proline to glutamate is a critical pathway in all organisms and holds roles in mental illness and cancer. The two enzymes utilized in the oxidation of proline are proline dehydrogenase (PRODH) and Δ1‐pyrroline‐5‐carboxylate dehydrogenase (P5CDH). In certain organisms, such as gram‐negative bacteria, these enzymes are fused into a single polypeptide named Proline Utilization A (PutA). In gram‐positive bacteria and eukaryotes, these enzymes exist as two separate polypeptides. A highly conserved active site lysine residue in PRODH enzymes has been shown by structural studies to be in proximity to the N1 hydrogen of proline. Previously, this lysine residue was proposed to deprotonate the N1 hydrogen of proline in Mycobacterium tuberculosis PRODH (MtPRODH), which is the first step in the mechanism of converting proline into glutamate (Serrano, H., and Blanchard, J. S., 2013. Biochemistry 2013, 52, 5009−5015). Substitution of this lysine residue with alanine eliminated catalytic activity in MtPRODH (Serrano, H., and Blanchard, J. S., 2013. Biochemistry 2013, 52, 5009−5015). To see if the role of this lysine is conserved in bifunctional PRODH/PutAs, we made the analogous mutation in PutA from Escherichia coli (EcPutA) and monofunctional PRODH from Thermus thermophilus (TtPRODH). Unexpectedly, this mutation only minimally affected the activity of EcPutA, whereas activity was completely lost by the mutation in TtPRODH, as observed previously for MtPRODH. These results indicate that there might be differences in the active site environments of monofunctional and bifunctional PRODH/PutAs