Development and retention of a primordial moonlighting pathway of protein modification in the absence of selection presents a puzzle

Abstract

Lipoic acid is synthesized by a remarkably atypical pathway in which the cofactor is assembled on its cognate proteins. An octanoyl moiety diverted from fatty acid synthesis is covalently attached to the acceptor protein, and sulfur insertion at carbons 6 and 8 of the octanoyl moiety form the lipoyl cofactor. Covalent attachment of this cofactor is required for function of several central metabolism enzymes, including the glycine cleavage H protein (GcvH). InBacillus subtilis, GcvH is the sole substrate for lipoate assembly. Hence lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. Lipoyl transfer has been argued to be the primordial pathway of OADH lipoylation. TheEscherichia colipathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to have arisen later. Because roughly 3 billion years separate the divergence of these bacteria, it is surprising thatE. coliGcvH functionally substitutes for theB. subtilisprotein in lipoyl transfer. Known and putative GcvHs from other bacteria and eukaryotes also substitute forB. subtilisGcvH in OADH modification. Because glycine cleavage is the primary GcvH role in ancestral bacteria that lack OADH enzymes, lipoyl transfer is a “moonlighting” function: that is, development of a new function while retaining the original function. This moonlighting has been conserved in the absence of selection by some, but not all, GcvH proteins. Moreover,Aquifex aeolicusencodes five putative GcvHs, two of which have the moonlighting function, whereas others function only in glycine cleavage.