Crystal structure of an HD‐GYP domain cyclic‐di‐GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre

Abstract

SummaryBis‐(3′,5′) cyclic di‐guanylate (c‐di‐GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c‐di‐GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD‐GYP domains. Here, we have determined the structure of an enzymatically active HD‐GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c‐di‐GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c‐di‐GMP. This structure completes the picture of all domains involved in c‐di‐GMP metabolism and reveals that the HD‐GYP family splits into two distinct subgroups containing bi‐ and trinuclear metal centres.

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

“…Strikingly, the structure shows a bimetallic active site whose metal binding mode is different from those of both Bd1817 and PmGH. Purified PA4781 did not contain iron in the active site as for previously reported HD-GYPs (14,16,18), and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 are in agreement with our previous data, indicating that this is preferentially a pGpG binding protein (15).…”

“…From a structural point of view, HD-GYPs are metal binding proteins named after their conserved motifs, but while the HD residues clearly serve as metal ligands, the role of the GYP triad is not so clear. Indeed, it was shown that mutation of the GYP signature renders the protein RpfG unable to interact with its specific GGDEF partners (12), while leaving the catalytic activity unaffected (14). Moreover, it should be recalled that it is not excluded that degenerated HDGYPs (i.e., mutated in the HD-GYP signature), though catalytically inactive, may still function as c-di-GMP or even pGpG receptors (15,16).…”

“…Recently, these puzzling issues benefited from the first two solved structures for this class of proteins. The first, Bd1817 (16), is a degenerated protein from Bdellovibrio bacteriovorus, while the second, PmGH, is an active enzyme from Persephonella marina (14). Both proteins were purified and crystallized as iron binding proteins; however, the two metal binding sites differ markedly, with Bd1817 showing a bi-iron center, while PmGH binds three iron ions with different oxidation states.…”

“…Both proteins were purified and crystallized as iron binding proteins; however, the two metal binding sites differ markedly, with Bd1817 showing a bi-iron center, while PmGH binds three iron ions with different oxidation states. Therefore, the authors of the latter structure suggested that the HD-GYP superfamily should be further divided in two subgroups, based on the presence of a bimetallic or a trimetallic center, and highlighted the need for further structural data to fully elucidate the features of this intriguing class of enzyme (14). We have recently presented a detailed biochemical characterization of two of the three HD-GYP proteins encoded by the genome of the human pathogen Pseudomonas aeruginosa (15), namely, PA4781 and PA4108, which were shown to affect c-di-GMP levels in vivo (the third, PA2572, displays a degenerated YN-GYP signature) (17).…”

An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis

“…This superfamily is divided into classes of enzymes that use a His-Asp doublet of residues and an additional series of conserved His and Asp residues to coordinate a single divalent metal (HX n HDX n D motif) (8,9), a dinuclear metal active site (HX n HDX n HX n HX n D motif) (10,11), or a recently described trinuclear iron metal-center (12). Although in many cases the identity of the relevant catalytic metal required for chemistry in vivo is unclear, there are examples of mononuclear enzymes that can use magnesium (13), cobalt (14), or manganese (8,13,15), and dinuclear enzymes that can use nickel (16), manganese (17), or iron (10,(18)(19)(20).…”