Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight

Abstract

5-Oxoproline (OP) is well-known as an enzymatic intermediate in the eukaryotic γ-glutamyl cycle, but it is also an unavoidable damage product formed spontaneously from glutamine and other sources. Eukaryotes metabolize OP via an ATP-dependent 5-oxoprolinase; most prokaryotes lack homologs of this enzyme (and the γ-glutamyl cycle) but are predicted to have some way to dispose of OP if its spontaneous formation is significant. Comparative analysis of prokaryotic genomes showed that the gene encoding pyroglutamyl peptidase, which removes N-terminal OP residues, clusters in diverse genomes with genes specifying homologs of a fungal lactamase (renamedrokaryotic 5-ooprolinase ,) and homologs of allophanate hydrolase subunits (renamed and). Inactivation of ,, or genes slowed growth, caused OP accumulation in cells and medium, and prevented use of OP as a nitrogen source. Assays of cell lysates showed that ATP-dependent 5-oxoprolinase activity disappeared when, , or was inactivated. 5-Oxoprolinase activity could be reconstituted by mixing recombinant PxpA, PxpB, and PxpC proteins. In addition, overexpressing genes in increased 5-oxoprolinase activity in lysates ≥1700-fold. This work shows that OP is a major universal metabolite damage product and that OP disposal systems are common in all domains of life. Furthermore, it illustrates how easily metabolite damage and damage-control systems can be overlooked, even for central metabolites in model organisms.

Structure–function relationships of the 5‐oxoprolinase subunit A: Guiding biological sciences students down the path less traveled

“…Subsequently, the 5‐oxoprolinase complex pxpA , pxpB , and pxpC genes, which occur in tandem, were experimentally determined to be co‐expressed and their gene products essential for conversion of 5‐oxoproline to L‐glutamate in both Bacillus subtilis and Escherichia coli . These findings formed the basis for the putative annotation of several 100 genes based on sequence homology with the 5‐oxoprolinase genes .…”

Enzyme promiscuity, metabolite damage, and metabolite damage control systems of the tricarboxylic acid cycle

“…However, the products of promiscuous enzymes and spontaneous chemical reactions are not always harmful. Buildup of some damaged metabolites has mild effects on fitness [8]. Promiscuous enzyme functions can also serve as the starting point for the evolution of new biochemical capabilities [119,120].…”

“…Enzymes can also occasionally catalyze an unintended reaction on a physiological substrate (i.e., catalytic promiscuity) [6,7]. Both substrate and catalytic promiscuities result in the formation of ‘unintended’, ‘noncanonical’, or ‘damaged’ metabolites (i.e., metabolite damage) that can be a useless drain on metabolism [8] and may be inhibitory [9], and/or reactive [10,11], sometimes leading to toxicity. In addition to enzyme promiscuity, spontaneous chemical reactions can occur to cellular metabolites causing metabolite damage [12].…”

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan–Lam Coupling

“…Pyroglutamate formation is catalyzed in nature by a glutaminyl cyclase (QC) enzyme . Pyroglutamate occurs commonly in natural proteins, but the role of pyroglutamate in living systems is not fully understood, presumably due in part to its subtle effect on local protein structure.…”