Molecular and structural characterization of agmatine coumaroyltransferase in Triticeae, the key regulator of hydroxycinnamic acid amide accumulation.

Hydroxycinnamic acid amides (HCAAs) are involved in stress-induced defense in many plant species. Barley accumulates high concentrations of HCAAs irrespective of exogenous stressors, while other major cereals such as wheat and rice accumulate relatively low levels of HCAAs in intact tissues. The primary HCAA species in barley are biosynthesized by agmatine p-coumaroyltransferase (ACT), an N-acyltransferase of the BAHD superfamily. However, the molecular basis underlying barley's uniquely high HCAA accumulation has not been elucidated, and information regarding the structural details of BAHD N-acyltransferases is limited. Hence, we aimed to investigate the ACTs of family Poaceae. We isolated ACT (-like) genes, including those previously undescribed, and investigated their enzymatic and genetic features. All the identified enzymes belonged to clade IVa of the BAHD superfamily. The barley and wheat ACTs were further categorized, based on catalytic properties and primary structures, into ACT1 and ACT2 groups, the encoding loci of which are neighbors on the same chromosome. While all ACTs exhibited similar Km values for CoA-thioesters (acyl-group donors), members of the ACT1 group showed a distinctly higher affinity for agmatine (acyl-acceptor). Among the ACTs tested, an ACT isozyme in barley (HvACT1-1) showed the highest catalytic efficiency and transcript level, indicating that ACT regulates high-level HCAA accumulation in barley. For further enzymatic characterization of the ACTs, we crystalized wheat ACT2 (TaACT2) and determined its structure at 2.3 Å resolution. Structural alignment of TaACT2 and HvACT1-1 showed that the architectures of the substrate binding pockets were well conserved. However, the structure of a loop located at the entrance to acyl-acceptor binding site may be more flexible in TaACT2, which could be responsible for the lower affinity of TaACT2 to agmatine. Mutations of HvACT1-1 at Glu372 and Asp374 within one of the clade-IV specific motifs facing the deduced acyl-acceptor binding pocket caused significant catalytic deterioration toward agmatine both in Km and kcat, suggesting their key roles in acyl acceptor binding by the clade-IV enzymes. This study elucidated the molecular basis of how plants accumulate defensive specialized metabolites and provided insights into developing efficient and eco-friendly agricultural methods.

Crystal structure of barley agmatine coumaroyltransferase, an N-acyltransferase from the BAHD superfamily.

The enzymes of the BAHD superfamily, a large group of acyl-CoA-dependent acyltransferases in plants, are involved in the biosynthesis of diverse secondary metabolites. While the structures of several O-acyltransferases from the BAHD superfamily, such as hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase, have been elucidated, no structural information on N-acyltransferases is available. Hordeum vulgare agmatine coumaroyltransferase (HvACT) is an N-acyltransferase from the BAHD superfamily and is one of the most important enzymes in the secondary metabolism of barley. Here, an apo-form structure of HvACT is reported as the first structure of an N-acyltransferase from the BAHD superfamily. HvACT crystals diffracted to 1.8 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 57.6, b = 59.5, c = 73.6 Å, α = 90, ß = 91.3 , γ = 90°. Like other known BAHD superfamily structures, HvACT contains two domains that adopt a two-layer αß-sandwich architecture and a solvent-exposed channel that penetrates the enzyme core.

Structural basis of substrate recognition by the substrate binding protein (SBP) of a hydrazide transporter, obtained from Microbacterium hydrocarbonoxydans.

Microbacterium hydrocarbonoxydans was isolated, using hydrazide compounds as its sole carbon source. The key enzyme that metabolizes these compounds was identified as hydrazidase, and the operon containing the gene coding for the enzyme, was revealed by genome sequencing. The operon also contained genes coding for an ATP-binding cassette transporter (ABC transporter), which was expected to transport the hydrazide compounds. Substrate binding protein (SBP), a component subunit of the transporter, plays an important role in recognizing the correct substrates for transport. Therefore, to elucidate the mechanism of recognition of the unnatural hydrazide compounds, we determined the crystal structures of the SBP, obtained from M. hydrocarbonoxydans (Mh-SBP), complexed with and without the hydrazide compound, at 2.2 Å and 1.75 Å resolutions, respectively. The overall structures of Mh-SBP were similar to those of the SBP in oligopeptide transporters such as OppA. On comparison, the liganded and unliganded structures of Mh-SBP showed an open - close conformation change. Interestingly, the binding mode of the compound to Mh-SBP was almost identical to that of the compound to hydrazidase, suggesting that the ABC transporter served transporting these compounds. Furthermore, based on the hydrazide complex structure, paraben, the other putative substrate of the protein, was successfully used with Mh-SBP to obtain the paraben complex structure.

Single amino acid mutation altered substrate specificity for L-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans.

scyllo-inositol dehydrogenase, isolated from Paracoccus laeviglucosivorans (Pl-sIDH), exhibits a broad substrate specificity: it oxidizes scyllo- and myo-inositols as well as L-glucose, converting L-glucose to L-glucono-1,5-lactone. Based on the crystal structures previously reported, Arg178 residue, located at the entry port of the catalytic site, seemed to be important for accepting substrates. Here, we report the role of Arg178 by using an alanine-substituted mutant for kinetic analysis as well as to determine the crystal structures. The wild-type Pl-sIDH exhibits the activity for scyllo-inositol most preferably followed by myo-inositol and L-glucose. On the contrary, the R178A mutant abolished the activities for both inositols, but remained active for L-glucose to the same extent as its wild-type. Based on the crystal structures of the mutant, the side chain of Asp191 flipped out of the substrate binding site. Therefore, Arg178 is important in positioning Asp191 correctly to exert its catalytic activities.Abbreviations: IDH: inositol dehydrogenase; LB: Luria-Bertani; kcat: catalyst rate constant; Km: Michaelis constant; NAD: nicotinamide dinucleotide; NADH: nicotinamide dinucleotide reduced form; PDB; Protein Data Bank; PDB entry: 6KTJ, 6KTK, 6KTL.

Structural basis for the substrate recognition of aminoglycoside 7''-phosphotransferase-Ia from Streptomyces hygroscopicus.

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7''-phosphotransferase-Ia [APH(7'')-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7'' and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5'-adenylyl-ß,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7'')-Ia and APH(4)-Ia, the crystal structure of APH(7'')-Ia complexed with HygB is reported. The overall structure of APH(7'')-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7'')-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7'')-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.

Crystal structure and kinetic analyses of a hexameric form of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum.

(S)-3-Hydroxybutyryl-CoA dehydrogenase (HBD) has been gaining increased attention recently as it is a key enzyme in the enantiomeric formation of (S)-3-hydroxybutyryl-CoA [(S)-3HB-CoA]. It converts acetoacetyl-CoA to (S)-3HB-CoA in the synthetic metabolic pathway. (S)-3HB-CoA is further modified to form (S)-3-hydroxybutyrate, which is a source of biodegradable polymers. During the course of a study to develop biodegradable polymers, attempts were made to determine the crystal structure of HBD from Clostridium acetobutylicum (CacHBD), and the crystal structures of both apo and NAD+-bound forms of CacHBD were determined. The crystals belonged to different space groups: P212121 and P21. However, both structures adopted a hexamer composed of three dimers in the asymmetric unit, and this oligomerization was additionally confirmed by gel-filtration column chromatography. Furthermore, to investigate the catalytic residues of CacHBD, the enzymatic activities of the wild type and of three single-amino-acid mutants were analyzed, in which the Ser, His and Asn residues that are conserved in the HBDs from C. acetobutylicum, C. butyricum and Ralstonia eutropha, as well as in the L-3-hydroxyacyl-CoA dehydrogenases from Homo sapiens and Escherichia coli, were substituted by alanines. The S117A and N188A mutants abolished the activity, while the H138A mutant showed a slightly lower Km value and a significantly lower kcat value than the wild type. Therefore, in combination with the crystal structures, it was shown that His138 is involved in catalysis and that Ser117 and Asn188 may be important for substrate recognition to place the keto group of the substrate in the correct position for reaction.

Small-molecule screening yields a compound that inhibits the cancer-associated transcription factor Hes1 via the PHB2 chaperone.

The transcription factor Hes family basic helix-loop-helix transcription factor 1 (Hes1) is a downstream effector of Notch signaling and plays a crucial role in orchestrating developmental processes during the embryonic stage. However, its aberrant signaling in adulthood is linked to the pathogenesis of cancer. In the present study, we report the discovery of small organic molecules (JI051 and JI130) that impair the ability of Hes1 to repress transcription. Hes1 interacts with the transcriptional corepressor transducing-like enhancer of split 1 (TLE1) via an interaction domain comprising two tryptophan residues, prompting us to search a chemical library of 1,800 small molecules enriched for indole-like π-electron-rich pharmacophores for a compound that blocks Hes1-mediated transcriptional repression. This screening identified a lead compound whose extensive chemical modification to improve potency yielded JI051, which inhibited HEK293 cell proliferation with an EC50 of 0.3 µm Unexpectedly, using immunomagnetic isolation and nanoscale LC-MS/MS, we found that JI051 does not bind TLE1 but instead interacts with prohibitin 2 (PHB2), a cancer-associated protein chaperone. We also found that JI051 stabilizes PHB2's interaction with Hes1 outside the nucleus, inducing G2/M cell-cycle arrest. Of note, JI051 dose-dependently reduced cell growth of the human pancreatic cancer cell line MIA PaCa-2, and JI130 treatment significantly reduced tumor volume in a murine pancreatic tumor xenograft model. These results suggest a previously unrecognized role for PHB2 in the regulation of Hes1 and may inform potential strategies for managing pancreatic cancer.

Crystallographic insights into the pore structures and mechanisms of the EutL and EutM shell proteins of the ethanolamine-utilizing microcompartment of Escherichia coli.

The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) of Escherichia coli is a polyhedral organelle that harbors specific enzymes for the catabolic degradation of ethanolamine. The compartment is composed of a proteinaceous shell structure that maintains a highly specialized environment for the biochemical reactions inside. Recent structural investigations have revealed hexagonal assemblies of shell proteins that form a tightly packed two-dimensional lattice that is likely to function as a selectively permeable protein membrane, wherein small channels are thought to permit controlled exchange of specific solutes. Here, we show with two nonisomorphous crystal structures that EutM also forms a two-dimensional protein membrane. As its architecture is highly similar to the membrane structure of EutL, it is likely that the structure represents a physiologically relevant form. Thus far, of all Eut proteins, only EutM and EutL have been shown to form such proteinaceous membranes. Despite their similar architectures, however, both proteins exhibit dramatically different pore structures. In contrast to EutL, the pore of EutM appears to be positively charged, indicating specificity for different solutes. Furthermore, we also show that the central pore structure of the EutL shell protein can be triggered to open specifically upon exposure to zinc ions, suggesting a specific gating mechanism.

Crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase from the hyperthermophile Thermotoga maritima for insights into the coordination of conformational changes and an inhibitor binding.

Isopentenyl diphosphate is a precursor of various isoprenoids and is produced by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids of plants, protozoa and many eubacteria. A key enzyme in the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be the target of fosmidomycin, which works as an antimalarial, antibacterial and herbicidal compound. In this paper, we report studies of kinetics and the crystal structures of the thermostable DXR from the hyperthermophile Thermotoga maritima. Unlike the mesophilic DXRs, Thermotoga DXR (tDXR) showed activity only with Mg(2+) at its growth temperature. We solved the crystal structures of tDXR with and without fosmidomycin. The structure without fosmidomycin but unexpectedly bound with 2-methyl-2,4-pentanediol (MPD), revealing a new extra space available for potential drug design. This structure adopted the closed form by rigid domain rotation but without the flexible loop over the active site, which was considered as a novel conformation. Further, the conserved Asp residue responsible for cation binding seemed to play an important role in adjusting the position of fosmidomycin. Taken together, our kinetic and the crystal structures illustrate the binding mode of fosmidomycin that leads to its slow, tight binding according to the conformational changes of DXR.