Two-step Ligand Binding in a (βα)8 Barrel Enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA*

HisA is a (βα)₈ barrel enzyme that catalyzes the Amadori rearrangement of N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) to N′-((5′-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the histidine biosynthesis pathway, and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo-state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp-7 acts as the catalytic base, and Asp-176 acts as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen bond network including His-47, Asp-129, Thr-171, and Ser-202, all shown to be functionally important. The closed conformation structure is highly similar to the bifunctional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.     

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr²¹³. The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.     

A eukaryotic LOV-histidine kinase with circadian clock function in the picoalga Ostreococcus

The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue‐light receptors such as aureochromes to absorb shorter‐wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV‐HK) that functions as a new class of eukaryotic blue‐light receptor in the pico‐phytoplanktonic cell Ostreococcus tauri. This LOV‐HK is related to the large family of LOV‐HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV‐HKs, including O. tauri LOV‐HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV‐HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV‐HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV‐HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O. tauri LOV‐HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O. tauriLOV‐HK alters CCA1 rhythmicity under constant red light, irrespective of LOV‐HK’s photochemical reactivity, suggesting that O. tauri LOV‐HK also participates in regulation of the circadian clock independent of its blue‐light‐sensing property. Molecular characterization of O. tauri LOV‐HK demonstrates that this type of photoreceptor family is not limited to prokaryotes.     

Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases

The bifunctional Escherichia coli glutathionylspermidine synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Its amidase domain (GspA), which catalyzes the hydrolysis of Gsp into glutathione and spermidine, plays an important role in redox sensing and protein S-thiolation. To gain insight of the regulation and catalytic mechanism of and further understand the recycling of the Gsp dimer and Gsp-S-protein adducts, we solved two crystal structures of GspA and GspSA both with the C59A mutation and bound with the substrate, Gsp. In both structures, Cys59, His131, and Glu147 form the catalytic triad, which is similar to other cysteine proteases. Comparison of the GspA_Gsp complex and apo GspSA structures indicates that on binding with Gsp, the side chains of Asn149 and Gln58 of the amidase domain are induced to move closer to the carbonyl oxygen of the cleaved amide bond of Gsp, thereby participating in catalysis. In addition, the helix-loop region of GspA, corresponding to the sequence (30)YSSLDPQEYEDDA(42), involves in regulating the substrate binding. Our previous study indicated that the thiol of Cys59 of GspA is only oxidized to sulfenic acid by H(2)O(2). When comparing the active site of GspA with those of other cysteine proteases, we found that limited space and hydrophobicity of the environment around Cys59 play an important role to inhibit its further oxidation. The structural results presented here not only elucidate the catalytic mechanism and regulation of GspA but also help us to design small molecules to inhibit or probe for the activity of GspA.     

Dimeric dUTPases, HisE, and MazG belong to a new superfamily of all-alpha NTP pyrophosphohydrolases with potential "house-cleaning" functions

Structure-guided analysis of the new dimeric dUTPase family revealed its sequence relationship to the phage T4 dCTPase, phosphoribosyl-ATP pyrophosphatase HisE, NTP pyrophosphatase MazG, and several uncharacterized protein families, including the human protein XTP3TPA (RS21-C6), which is overexpressed in embryonic and cancer cells. Comparison with the recently determined structure of a MazG-like protein from Sulfolobus solfataricus supported the unification of these enzymes in one superfamily of all-alpha NTP pyrophosphatases, suggesting that dimeric dUTPases evolved from a tetrameric MazG-like ancestor by gene duplication. Analysis of the structure of the Sulfolobus MazG points to 2-hydroxyadenosine (isoguanosine) triphosphate, a product of oxidative damage of ATP, as the most likely substrate. We predict that uncharacterized members of this superfamily perform "house-cleaning" functions by hydrolyzing abnormal NTPs and are functionally analogous to the structurally unrelated hydrolases of the Nudix superfamily. We outline probable tertiary and quaternary structures of the all-alpha NTP pyrophosphatase superfamily members.     

Effect of exogenous histidine on restoration of electron transfer on the donor side of Photosystem II depleted of Mn

It is shown that restoration of photoinduced electron flow with added Mn²⁺ (measured by photoreduction of DCPIP and photoinduced change of chlorophyll fluorescence yield) in Mn-depleted Photosystem II (PS II) membrane fragments isolated from spinach chloroplasts, is considerably increased by exogenous histidine (His). The stimulating effect of His is not observed if other electron donors (NH₂OH or diphenylcarbazide) are used instead of Mn²⁺. His added alone does not induce electron transfer in Mn-depleted PS II preparations. Investigation of pH dependence of the stimulating effect of 2 mM His shows that the effect is observed only at pH > 5.0, it gives a 50% activation around pH 6.0 and saturates at pH 7.0–7.5. Nearly 200 μM His is required for a 50 effect at pH 7.0. It is suggested that the added His can be involved in stimulation of electron transfer on the donor side of PS II through direct interaction of Mn²⁺ with deprotonated form(s) of His resulting in formation of Mn–His complexes capable of efficient electron donation to PS II (though it is not excluded that His serves as a base that takes part in proton exchange coupled with redox reactions on the donor side of PS II or as an electron donor to the oxidized Mn).     

Highly branched HK peptides are effective carriers of siRNA

BACKGROUND: Both viral and nonviral carriers have been used to carry small interfering RNA molecules (siRNA) to their cytosolic mRNA target. To date, few peptide carriers have been developed that have proved effective for siRNA delivery. Our previous branched carriers composed of histidine and lysine were useful for transfection of plasmids. In this study, we determined if these and more highly branched HK polymers were effective carriers of siRNA. METHODS: Several branched polymers were synthesized on a Ranin Voyager synthesizer. These polymers were then screened for their ability to transfer siRNA into SVR-bag4 cells, MDA-MB-435 cells, and C6 cells. After one polymer, H3K8b, was identified as an effective carrier of siRNA, additional polymers were synthesized to determine the essential domains for siRNA transport. The size/zeta-potential of HK : siRNA complexes were measured with the N4 submicron particle size analyzer and the Delsa 440 SX zeta-potential analyzer, respectively. Toxicity of the highly branched polymers in complex with siRNA was investigated by flow cytometry. RESULTS: In an endothelial cell line (SVR-bag4) that stably expressed beta-galactosidase (beta-gal), an siRNA in complex with the H3K8b polymer inhibited beta-gal expression by more than 80%. In contrast, the polymer H2K4b, which was an effective carrier of plasmids, was not an efficient carrier of siRNA. The size and surface charge did not distinguish effective from ineffective HK carriers of siRNA. By modifying H3K8b, we then determined what properties of H3K8b augmented siRNA delivery. The histidine-rich domain and the length of the terminal arms of H3K8 were important for siRNA delivery. The modestly more effective analog of H3K8b containing an integrin ligand, H3K8b(+RGD), was able to inhibit markedly intracellular beta-gal expression. Furthermore, we determined that H3K8b(+RGD) in complex with a luciferase-targeting siRNA inhibited luciferase expression in MDA-MB-435 cells. At its optimal concentration for inhibiting its target, H3K8b(+RGD) : siRNA complex had minimal toxicity. In contrast, carriers of siRNA such as Oligofectamine and Lipofectamine 2000 were significantly more toxic. CONCLUSIONS: Both the degree of complexity and the sequence specificity are important factors to be considered for developing the HK carrier of siRNA. In particular, we found that certain branched HK polymers (H3K8b, H3K8b(+RGD), and similar structural analogs) with eight terminal branches and a histidine-rich domain were effective carriers of siRNA.     

Selection and Characterization of Nickel-Tolerant Tobacco Cells

Tobacco (Nicotiana tabacum L. cv. BY-2) cell lines tolerant to 700 M Ni in which unselected cells can not grow, were selected. The Ni-tolerant cells were also more tolerant to Co, but not to Cd than unselected cells. Ni concentrations in Ni-tolerant cells were always higher than those in medium. Since buthionine sulfoximine did not affect their Ni-tolerance, it is suggested that phytochelatins are not involved in Ni-tolerance of Ni-tolerant cells. On the other hand, histidine contents in Ni-tolerant and unselected cells, which were treated with Ni, were higher that those treated without Ni, and the degree of the elevation of histidine contents by Ni-treatment was higher in Ni-tolerant cells than in unselected cells. Additionally, exogenous histidine reduced the inhibitory effect of Ni on the growth of unselected cells. In addition, the cells that were tolerant to histidine-analogue, had higher contents of histidine and Ni-tolerance. These results suggest that histidine is involved in Ni-tolerance and the detoxification of Ni in symplast in Ni-tolerant cells.     

Structure and action of urocanase

Urocanase (EC 4.2.1.49) from Pseudomonas putida was crystallized after removing one of the seven free thiol groups. The crystal structure was solved by multiwavelength anomalous diffraction (MAD) using a seleno-methionine derivative and then refined at 1.14 A resolution. The enzyme is a symmetric homodimer of 2 x 557 amino acid residues with tightly bound NAD+ cofactors. Each subunit consists of a typical NAD-binding domain inserted into a larger core domain that forms the dimer interface. The core domain has a novel chain fold and accommodates the substrate urocanate in a surface depression. The NAD domain sits like a lid on the core domain depression and points with the nicotinamide group to the substrate. Substrate, nicotinamide and five water molecules are completely sequestered in a cavity. Most likely, one of these water molecules hydrates the substrate during catalysis. This cavity has to open for substrate passage, which probably means lifting the NAD domain. The observed atomic arrangement at the active center gives rise to a detailed proposal for the catalytic mechanism that is consistent with published chemical data. As expected, the variability of the residues involved is low, as derived from a family of 58 proteins annotated as urocanases in the data banks. However, one well-embedded member of this family showed a significant deviation at the active center indicating an incorrect annotation.     

脆性组氨酸三联体基因小干扰RNA的构建及干扰效果检测

目的:构建脆性组氨酸三联体(FHIT)基因的小干扰RNA(siRNA),并检测其对293T细胞内源性FHIT基因表达的干扰效果。方法:根据FHIT基因序列,通过生物信息学网站预测可能的siRNA,从中筛选出2条合理的FHIT-siRNA,将其克隆到siRNA表达载体pSilencer2.1-U6Hygro中,转化大肠杆菌DH5α,挑取阳性克隆进行测序鉴定;将构建成功的FHIT-siRNA重组载体转染人胚肾细胞293T,Western印迹检测siRNA对293T内源性FHIT表达的干扰效果。结果:构建了2个FHIT-siRNA重组质粒,2条siRNA都有干扰作用,其中一条的作用效果可达50%以上。结论:构建的FHIT-siRNA为进一步研究FHIT的功能提供了有利的工具。