Responsiveness of sphingosine phosphate lyase insufficiency syndrome to vitamin B6 cofactor supplementation.

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamin's chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation.

Crystal structures reveal a new and novel FoxO1 binding site within the human glucose-6-phosphatase catalytic subunit 1 gene promoter.

Human glucose-6-phosphatase plays a vital role in blood glucose homeostasis and holds promise as a therapeutic target for diabetes. Expression of its catalytic subunit gene 1 (G6PC1) is tightly regulated by metabolic-response transcription factors such as FoxO1 and CREB. Although at least three potential FoxO1 binding sites (insulin response elements, IREs) and one CREB binding site (cAMP response element, CRE) within the proximal region of the G6PC1 promoter have been identified, the interplay between FoxO1 and CREB and between FoxO1 bound at multiple IREs has not been well characterized. Here we present the crystal structures of the FoxO1 DNA binding domain in complex with the G6PC1 promoter. These complexes reveal the presence of a new non-consensus FoxO1 binding site that overlaps the CRE, suggesting a mutual exclusion mechanism for FoxO1 and CREB binding at the G6PC1 promoter. Additional findings include (i) non-canonical FoxO1 recognition sites, (ii) incomplete FoxO1 occupancies at the available IRE sites, and (iii) FoxO1 dimeric interactions that may play a role in stabilizing DNA looping. These findings provide insight into the regulation of G6PC1 gene transcription by FoxO1, and demonstrate a high versatility of target gene recognition by FoxO1 that correlates with its diverse roles in biology.

Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA(DeltaEFG).

Complex enzymes containing Fe-S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe-4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear. Here we show the structure of HydA(DeltaEFG) (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe-4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe-4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA.

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²âº. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.

FAD binding by ApbE protein from Salmonella enterica: a new class of FAD-binding proteins.

The periplasmic protein ApbE was identified through the analysis of several mutants defective in thiamine biosynthesis and was implicated as having a role in iron-sulfur cluster biosynthesis or repair. While mutations in apbE cause decreased activity of several iron-sulfur enzymes in vivo, the specific role of ApbE remains unknown. Members of the AbpE family include NosX and RnfF, which have been implicated in oxidation-reduction associated with nitrous oxide and nitrogen metabolism, respectively. In this work, we show that ApbE binds one FAD molecule per monomeric unit. The structure of ApbE in the presence of bound FAD reveals a new FAD-binding motif. Protein variants that are nonfunctional in vivo were generated by random and targeted mutagenesis. Each variant was substituted in the environment of the FAD and analyzed for FAD binding after reconstitution. The variant that altered a key tyrosine residue involved in FAD binding prevented reconstitution of the protein.

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes.

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non-polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild-type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate-analog bound conformation and the third hinge angle is intermediate to the other two. The active-site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active-site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active-site conformations.

Human CTP synthase filament structure reveals the active enzyme conformation.

The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we show that for human CTPS, polymerization increases catalytic activity. The cryo-EM structures of bacterial and human CTPS filaments differ considerably in overall architecture and in the conformation of the CTPS protomer, explaining the divergent consequences of polymerization on activity. The structure of human CTPS filament, the first structure of the full-length human enzyme, reveals a novel active conformation. The filament structures elucidate allosteric mechanisms of assembly and regulation that rely on a conserved conformational equilibrium. The findings may provide a mechanism for increasing human CTPS activity in response to metabolic state and challenge the assumption that metabolic filaments are generally storage forms of inactive enzymes. Allosteric regulation of CTPS polymerization by ligands likely represents a fundamental mechanism underlying assembly of other metabolic filaments.

Getting a handle on the role of coenzyme M in alkene metabolism.

Coenzyme M (2-mercaptoethanesulfonate; CoM) is one of several atypical cofactors discovered in methanogenic archaea which participate in the biological reduction of CO(2) to methane. Elegantly simple, CoM, so named for its role as a methyl carrier in all methanogenic archaea, is the smallest known organic cofactor. It was thought that this cofactor was used exclusively in methanogenesis until it was recently discovered that CoM is a key cofactor in the pathway of propylene metabolism in the gram-negative soil microorganism Xanthobacter autotrophicus Py2. A four-step pathway requiring CoM converts propylene and CO(2) to acetoacetate, which feeds into central metabolism. In this process, CoM is used to activate and convert highly electrophilic epoxypropane, formed from propylene epoxidation, into a nucleophilic species that undergoes carboxylation. The unique properties of CoM provide a chemical handle for orienting compounds for site-specific redox chemistry and stereospecific catalysis. The three-dimensional structures of several of the enzymes in the pathway of propylene metabolism in defined states have been determined, providing significant insights into both the enzyme mechanisms and the role of CoM in this pathway. These studies provide the structural basis for understanding the efficacy of CoM as a handle to direct organic substrate transformations at the active sites of enzymes.

Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase.

The structures of beta class carbonic anhydrases (beta-CAs) determined so far fall into two distinct subclasses based on the observed coordination of the catalytic zinc (Zn2+) ion. The subclass of beta-CAs that coordinate Zn2+ tetrahedrally with four protein-derived ligands is represented by the structures of orthologues from Porphyridium purpureum, Escherichia coli, and Mycobacterium tuberculosis. Here we present the structure of an additional member of that subclass, that from Haemophilus influenzae, as well as detailed kinetic analysis, revealing the correspondence between structural classification and kinetic profile for this subclass. In addition, we identify a unique, noncatalytic binding mode for the substrate bicarbonate that occurs in both the H. influenzae and E. coli enzymes. The kinetic and structural analysis indicates that binding of bicarbonate in this site of the enzyme may modulate its activity by influencing a pH-dependent, cooperative transition between active and inactive forms. We hypothesize that the two structural subclasses of beta-CAs may provide models for the proposed active and inactive forms of the H. influenzae and E. coli enzymes.

Mechanisms of product feedback regulation and drug resistance in cytidine triphosphate synthetases from the structure of a CTP-inhibited complex.

Cytidine triphosphate synthetases (CTPSs) synthesize CTP and regulate its intracellular concentration through direct interactions with the four ribonucleotide triphosphates. In particular, CTP product is a feedback inhibitor that competes with UTP substrate. Selected CTPS mutations that impart resistance to pyrimidine antimetabolite inhibitors also relieve CTP inhibition and cause a dramatic increase in intracellular CTP concentration, indicating that the drugs act by binding to the CTP inhibitory site. Resistance mutations map to a pocket that, although adjacent, does not coincide with the expected UTP binding site in apo Escherichia coli CTPS [EcCTPS; Endrizzi, J. A., et al. (2004) Biochemistry 43, 6447-6463], suggesting allosteric rather than competitive inhibition. Here, bound CTP and ADP were visualized in catalytically active EcCTPS crystals soaked in either ATP and UTP substrates or ADP and CTP products. The CTP cytosine ring resides in the pocket predicted by the resistance mutations, while the triphosphate moiety overlaps the putative UTP triphosphate binding site, explaining how CTP competes with UTP while CTP resistance mutations are acquired without loss of catalytic efficiency. Extensive complementarity and interaction networks at the interfacial binding sites provide the high specificity for pyrimidine triphosphates and mediate nucleotide-dependent tetramer formation. Overall, these results depict a novel product inhibition strategy in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate subsites. This arrangement allows for independent adaptation of UTP and CTP binding affinities while efficiently utilizing the enzyme surface.

Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets.

Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.

Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases.

A family of eukaryotic-like Ser/Thr protein kinases occurs in bacteria, but little is known about the structures and functions of these proteins. Here we characterize PknB, a transmembrane signaling kinase from Mycobacterium tuberculosis. The intracellular PknB kinase domain is active autonomously, and the active enzyme is phosphorylated on residues homologous to regulatory phospho-acceptors in eukaryotic Ser/Thr kinases. The crystal structure of the PknB kinase domain in complex with an ATP analog reveals the active conformation. The predicted fold of the PknB extracellular domain matches the proposed targeting domain of penicillin-binding protein 2x. The structural and chemical similarities of PknB to metazoan homologs support a universal activation mechanism of Ser/Thr protein kinases in prokaryotes and eukaryotes.