Dynamics and Stabilization of the Human Gut Microbiome during the First Year of Life.

Phosphatases of the haloacid dehalogenase (HAD) superfamily of hydrolases are an ancient and very large class of enzymes that have evolved to dephosphorylate a wide range of low- and high molecular weight substrates with often exquisite specificities. HAD phosphatases constitute approximately one-fifth of all human phosphatase catalytic subunits. While the overall sequence similarity between HAD phosphatases is generally very low, family members can be identified based on the presence of a characteristic Rossmann-like fold and the active site sequence DxDx(V/T). HAD phosphatases employ an aspartate residue as a nucleophile in a magnesium-dependent phosphoaspartyl transferase reaction. Although there is genetic evidence demonstrating a causal involvement of some HAD phosphatases in diseases such as cancer, cardiovascular, metabolic and neurological disorders, the physiological roles of many of these enzymes are still poorly understood. In this review, we discuss the structure and evolution of human HAD phosphatases, and summarize their known functions in health and disease.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1742-4658.2012.08633.x

Human HAD phosphatases: structure, mechanism, and roles in health and disease.

Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.

https://www.pnas.org/content/pnas/112/16/E1974.full.pdf

Panoramic view of a superfamily of phosphatases through substrate profiling

Lipin proteins and glycerolipid metabolism: Roles at the ER membrane and beyond.

Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.

Human lipin1 catalyzes the highly regulated conversion of phosphatidic acids to diacylglycerides. Lipin’s cellular location, protein partners, and biological function are directed by phosphorylation−dephosphorylation events catalyzed by the phosphoserine phosphatase dullard. To define the determinants of dullard substrate recognition and catalysis, and hence, lipin regulation, steady-state kinetic analysis was performed on phosphoserine-bearing nonapeptides based on the phosphorylation sites of lipin. The results demonstrate that dullard shows specificity for the peptide corresponding to the insulin-dependent phosphorylation site (Ser106) of lipin with a kcat/Km of 2.9 × 104 M−1 s−1. These results are consistent with a coil−loop structure for the insulin-dependent phosphorylation site on human lipin1 and make unlikely the requirement for an adaptor protein to confer activity such as that proposed for the yeast homologue.

Homo sapiens dullard protein phosphatase shows a preference for the insulin-dependent phosphorylation site of lipin1.

The hotdog-fold superfamily is a functionally diverse family of evolutionarily related enzymes, which share a common α + β-fold. The founding member of this superfamily is the Escherichia coli β-hydroxydecanoyl-holo acyl carrier protein (ACP) dehydrase/isomerase.a5 Smith and co-workers coined the name “hotdog-fold” to represent the observed tertiary structure consisting of a 5-turn α-helix cradled by a curved, 7-stranded antiparallel β-sheet. The functional unit was reported as a dimer, with the subunit interface joining the two sheets into a continuous 14-stranded β-sheet. The two active sites are located at opposite ends of the interfaced sheets.

Soon after the report of the dehydratase/isomerase, the hotdog-fold was observed in the 4-hydroxybenzoyl-coenzyme A thioesterase from Pseudomonas sp. strain CBS3 (Ps 4-HB-CoA TE)1 and shortly thereafter in the 4-HB-CoA thioesterase from Arthrobacter special strain AU (Ar 4-HB-CoA TE)3 (Figure ​(Figure1A1A and B). Both thioesterases catalyze the final step of the 4-chlorobenzoate-degradation pathway.6,7 Surprisingly, the two thioesterases share little sequence identity (<25%), and although the tertiary structures are alike, the arrangements of the dimers, which form the respective (biological unit) tetramers, differ. Specifically, the dimers of the Ar 4-HB-CoA TE associate β-sheet-to-β-sheet, with the active sites directed to solvent, whereas in the Ps 4-HB-CoA TE structure the dimers are rotated 180° such that the active sites are directed to the dimer–dimer interface.1−3



Figure 1

(A) Tetrameric structure (dimer of dimers) of the hotdog-fold superfamily clade AA member 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas sp. strain CBS3 represented in Pymol.2 The two structural representations are related by a 90° vertical rotation. ...



Most relevant to the catalytic mechanism is the fact that even though the secondary structural elements that form the active sites of the two thioesterases are highly conserved, the constellation of catalytic residues is dissimilar as is the spatial location of the essential catalytic carboxylate residue.2,3 Based on the noted structural differences, the Ar 4-HB-CoA TE and Ps 4-HB-CoA TE are viewed as prototypes of two evolutionarily distinct clades of thioesterases within the hotdog-fold superfamily.b Notably, the common fold that links these two structural clades dictates that, for all members, substrate recognition is directed at holoACP or CoA-based thioester metabolites.

The thioesterases constitute a large subfamily within the hotdog-fold superfamily. The biological function of the thioesterase is defined by the identity of its in vivo substrate(s) (i.e., physiological substrate(s)) in combination with the biochemical context, as determined by protein partners and/or elements of regulation. When subjected to an in vitro activity screen, the typical hotdog-fold thioesterase displays hydrolytic activities toward a range of thioesters that extend well beyond the physiological substrate(s).8−12 Whereas this lack of substrate specificity might, in earlier times, have been interpreted as evidence for an “under-evolved” enzyme (befitting the role of “scavenger”), it is now well recognized that substrate promiscuity is a key trait of highly evolvable enzymes (for review, see ref (13)). We posit that this trait is inherent in the simplicity and plasticity of the architecture of the hotdog-fold.

Presently, we have carried out a case study of two paralogs, namely, YdiI and YbdB from E. coli. These thioesterases are related by 59% sequence identity and are distinguished by very diverse biological functions as detailed in the preceding companion paper14 and depicted in Scheme 1. Briefly, in E. coli, YdiI participates in menaquinone biosynthesis as a catalyst for the hydrolysis of the pathway intermediate, 1,4-dihydroxynapthoyl-CoA.14−16 YbdB, on the other hand, assists in the biosynthesis of the siderophore enterobactin through catalyzed hydrolysis of aberrant aroyl-holoEntB thioesters that, as dead-end pathway intermediates, act to block enterobactin formation.17−19



Scheme 1

Biological Functions of E. coli YdiI and YbdB



Herein, the structural determinants for substrate recognition and catalysis in these two paralogs are reported, and analyzed, within the context of evolution of hotdog-fold thioesterase function. In addition, because YdiI and YbdB are members of the same family clade as Ar 4-HB-CoA TE (Figure ​(Figure1B1B and C),3 a comparison of the relevant elements of structure and mechanism with those of this evolutionarily more distant relative is made, to identify those elements, which have been retained despite extensive sequence divergence. The findings are interpreted to suggest that a phosphopantethiene binding site is conserved for targeting CoA and holoACP-based thioester metabolites, and that the location of the ACP or adenosine 3′,5′-diphosphate binding site on the surface of the thioesterase serves to promote promiscuity toward these two classes of thioesters. Furthermore, we infer that the divergence in the alkyl/aryl binding site sequence is important for the evolution of new biological function. Finally, we propose that the roles played by the conserved catalytic residues in thioester hydrolysis change in the process of adaptation of the thioesterase to its new physiological substrate.

https://pubs.acs.org/doi/pdf/10.1021/bi500334v

Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2013.07.009

Co‐evolution of HAD phosphatase and hotdog‐fold thioesterase domain function in the menaquinone‐pathway fusion proteins BF1314 and PG1653

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Homo sapiens dullard protein phosphatase shows a preference for the insulin-dependent phosphorylation site of lipin1.

Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB

Co‐evolution of HAD phosphatase and hotdog‐fold thioesterase domain function in the menaquinone‐pathway fusion proteins BF1314 and PG1653