UDP‐Api/UDP‐Xyl synthases affect plant development by controlling the content of UDP‐Api to regulate the RG‐II‐borate complex

Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development.     

Functional and evolutionary analysis of viral proteins containing a Rossmann‐like fold

Viruses are the most abundant life form and infect practically all organisms. Consequently, these obligate parasites are a major cause of human suffering and economic loss. Rossmann‐like fold is the most populated fold among α/β‐folds in the Protein Data Bank and proteins containing Rossmann‐like fold constitute 22% of all known proteins 3D structures. Thus, analysis of viral proteins containing Rossmann‐like domains could provide an understanding of viral biology and evolution as well as could propose possible targets for antiviral therapy. We provide functional and evolutionary analysis of viral proteins containing a Rossmann‐like fold found in the evolutionary classification of protein domains (ECOD) database developed in our lab. We identified 81 protein families of bacterial, archeal, and eukaryotic viruses in light of their evolution‐based ECOD classification and Pfam taxonomy. We defined their functional significance using enzymatic EC number assignments as well as domain‐level family annotations.     

Crystal structures of halohydrin hydrogen‐halide‐lyases from Corynebacterium sp. N‐1074

Halohydrin hydrogen‐halide‐lyase (H‐Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2‐diol. Until now, six different H‐Lyases have been studied. These H‐Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N‐1074 has two different isozymes of H‐Lyase, HheA (A‐type) and HheB (B‐type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H‐Lyases. Among the B‐type H‐Lyases, HheB shows relatively high enantioselectivity compared to that of HheB_GP1. This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3‐dicyano‐2‐propanol at the catalytic site in the crystal structure of the HheB‐DiCN complex suggests that the product should be (R)‐epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity. Proteins 2015; 83:2230–2239. © 2015 Wiley Periodicals, Inc.     

Crystal structure of ADP‐dependent glucokinase from Methanocaldococcus jannaschii in complex with 5‐iodotubercidin reveals phosphoryl transfer mechanism

ADP‐dependent glucokinase (ADPGK) is an alternative novel glucose phosphorylating enzyme in a modified glycolysis pathway of hyperthermophilic Archaea. In contrast to classical ATP‐dependent hexokinases, ADPGK utilizes ADP as a phosphoryl group donor. Here, we present a crystal structure of archaeal ADPGK from Methanocaldococcus jannaschii in complex with an inhibitor, 5‐iodotubercidin, d ‐glucose, inorganic phosphate, and a magnesium ion. Detailed analysis of the architecture of the active site allowed for confirmation of the previously proposed phosphorylation mechanism and the crucial role of the invariant arginine residue (Arg197). The crystal structure shows how the phosphate ion, while mimicking a β‐phosphate group, is positioned in the proximity of the glucose moiety by arginine and the magnesium ion, thus providing novel insights into the mechanism of catalysis. In addition, we demonstrate that 5‐iodotubercidin inhibits human ADPGK‐dependent T cell activation‐induced reactive oxygen species (ROS) release and downstream gene expression, and as such it may serve as a model compound for further screening for hADPGK‐specific inhibitors.     

Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly

Archaeal flagella are unique structures that share functional similarity with bacterial flagella, but are structurally related to bacterial type IV pili. The flagellar accessory protein FlaH is one of the conserved components of the archaeal motility system. However, its function is not clearly understood. Here, we present the 2.2 Å resolution crystal structure of FlaH from the hyperthermophilic archaeon, Methanocaldococcus jannaschii. The protein has a characteristic RecA‐like fold, which has been found previously both in archaea and bacteria. We show that FlaH binds to immobilized ATP—however, it lacks ATPase activity. Surface plasmon resonance analysis demonstrates that ATP affects the interaction between FlaH and the archaeal motor protein FlaI. In the presence of ATP, the FlaH‐FlaI interaction becomes significantly weaker. A database search revealed similarity between FlaH and several DNA‐binding proteins of the RecA superfamily. The closest structural homologs of FlaH are KaiC‐like proteins, which are archaeal homologs of the circadian clock protein KaiC from cyanobacteria. We propose that one of the functions of FlaH may be the regulation of archaeal motor complex assembly.     

Crystal stuctures of MglB‐2 (TP0684), a topologically variant d‐glucose‐binding protein from Treponema pallidum,reveal a ligand‐induced conformational change

Previously, we determined the crystal structure of apo‐TpMglB‐2, a d ‐glucose‐binding component of a putative ABC transporter from the syphilis spirochete Treponema pallidum. The protein had an unusual topology for this class of proteins, raising the question of whether the d ‐glucose‐binding mode would be different in TpMglB‐2. Here, we present the crystal structures of a variant of TpMglB‐2 with and without d ‐glucose bound. The structures demonstrate that, despite its aberrant topology, the protein undergoes conformational changes and binds d ‐glucose similarly to other Mgl‐type proteins, likely facilitating d ‐glucose uptake in T. pallidum.     

Crystal structure of decaprenylphosphoryl‐β‐ D‐ribose 2'‐epimerase from Mycobacterium smegmatis

Decaprenylphosphoryl‐β‐D ‐ribose 2'‐epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti‐tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C‐terminus and shares homology with FAD‐dependent oxidoreductases of the vanillyl‐alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Phylogenomic analysis of UDP‐dependent glycosyltransferases provides insights into the evolutionary landscape of glycosylation in plant metabolism

Glycosylated metabolites generated by UDP‐dependent glycosyltransferases (UGTs) play critical roles in plant interactions with the environment as well as human and animal nutrition. The evolution of plant UGTs has previously been explored, but with a limited taxon sampling. In this study, 65 fully sequenced plant genomes were analyzed, and stringent criteria for selection of candidate UGTs were applied to ensure a more comprehensive taxon sampling and reliable sequence inclusion. In addition to revealing the overall evolutionary landscape of plant UGTs, the phylogenomic analysis also resolved the phylogenetic association of UGTs from free‐sporing plants and gymnosperms, and identified an additional UGT group (group R) in seed plants. Furthermore, lineage‐specific expansions and contractions of UGT groups were detected in angiosperms, with the total number of UGTs per genome remaining constant generally. The loss of group Q UGTs in Poales and Brassicales, rather than functional convergence in the group Q containing species, was supported by a gene tree of group Q UGTs sampled from many species, and further corroborated by the absence of group Q homologs on the syntenic chromosomal regions in Arabidopsis thaliana (Brassicales). Branch‐site analyses of the group Q UGT gene tree allowed for identification of branches and amino acid sites that experienced episodic positive selection. The positively selected sites are located on the surface of a representative group Q UGT (PgUGT95B2), away from the active site, suggesting their role in protein folding/stability or protein–protein interactions.     

Heating‐induced conformational change of a novel β‐(1→3)‐D‐glucan from Pleurotus geestanus

Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical–chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel β‐(1→3)‐D ‐glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical β‐(1→3)‐D ‐glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8–12.5°C and 25–60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro‐differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating‐induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating‐induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121–131, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Crystal structure of AFV1‐102, a protein from the acidianus filamentous virus 1

Viruses infecting hyperthermophilic archaea have intriguing morphologies and genomic properties. The vast majority of their genes do not have homologs other than in other hyperthermophilic viruses, and the biology of these viruses is poorly understood. As part of a structural genomics project on the proteins of these viruses, we present here the structure of a 102 amino acid protein from acidianus filamentous virus 1 (AFV1‐102). The structure shows that it is made of two identical motifs that have poor sequence similarity. Although no function can be proposed from structural analysis, tight binding of the gateway tag peptide in a groove between the two motifs suggests AFV1‐102 is involved in protein protein interactions.     

The three‐dimensional structure of “Lonely Guy” from Claviceps purpurea provides insights into the phosphoribohydrolase function of Rossmann fold‐containing lysine decarboxylase‐like proteins

The recently discovered cytokinin (CK)‐specific phosphoribohydrolase “Lonely Guy” (LOG) is a key enzyme of CK biosynthesis, converting inactive CK nucleotides into biologically active free bases. We have determined the crystal structures of LOG from Claviceps purpurea (cpLOG) and its complex with the enzymatic product phosphoribose. The structures reveal a dimeric arrangement of Rossmann folds, with the ligands bound to large pockets at the interface between cpLOG monomers. Structural comparisons highlight the homology of cpLOG to putative lysine decarboxylases. Extended sequence analysis enabled identification of a distinguishing LOG sequence signature. Taken together, our data suggest phosphoribohydrolase activity for several proteins of unknown function. Proteins 2015; 83:1539–1546. © 2015 Wiley Periodicals, Inc.     

Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin,glycyrrhizin

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins.     

Crystal structure of the ubiquitin‐like small archaeal modifier protein 2 from Haloferax volcanii

The discovery of ubiquitin‐like small archaeal modifier protein 2 (SAMP2) that forms covalent polymeric chains in Haloferax volcanii has generated tremendous interest in the function and regulation of this protein. At present, it remains unclear whether the Hfx. volcanii modifier protein SAMP1 has such polyubiquitinating‐like activity. Although SAMP1 and SAMP2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. To better understand the mechanism of SAMP2 conjugation, we have sought to characterize the biophysical and structural properties of the protein from Hfx. volcanii. SAMP2 is only partially structured under mesohalic solution conditions and adopts a well‐folded compact conformation in the presence of 2.5M of NaCl. Its 2.3‐Å‐resolution crystal structure reveals a characteristic α/β central core domain and a unique β‐hinge motif. This motif anchors an unusual C‐terminal extension comprising the diglycine tail as well as two lysine residues that can potentially serve to interlink SAMP2 moieties. Mutational alternation of the structural malleability of this β‐hinge motif essentially abolishes the conjugation activity of SAMP2 in vivo. In addition, NMR structural studies of the putative ubiquitin‐like protein HVO_2177 from Hfx. volcanii show that like SAMP1, HVO_2177 forms a classic β‐grasp fold in a salt‐independent manner. These results provide insights into the structure–function relationship of sampylating proteins of fundamental importance in post‐translational protein modification and environmental cues in Archaea.     

Heparin‐induced amyloid fibrillation of β2‐microglobulin explained by solubility and a supersaturation‐dependent conformational phase diagram

Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative and classical view of amyloid fibrillation: amyloid fibrils are crystal‐like precipitates of denatured proteins formed above solubility upon breaking supersaturation. Various additives accelerate and then inhibit amyloid fibrillation in a concentration‐dependent manner, suggesting that the combined effects of stabilizing and destabilizing forces affect fibrillation. Heparin, a glycosaminoglycan and anticoagulant, is an accelerator of fibrillation for various amyloidogenic proteins. By using β₂‐microglobulin, a protein responsible for dialysis‐related amyloidosis, we herein examined the effects of various concentrations of heparin on fibrillation at pH 2. In contrast to previous studies that focused on accelerating effects, higher concentrations of heparin inhibited fibrillation, and this was accompanied by amorphous aggregation. The two‐step effects of acceleration and inhibition were similar to those observed for various salts. The results indicate that the anion effects caused by sulfate groups are one of the dominant factors influencing heparin‐dependent fibrillation, although the exact structures of fibrils and amorphous aggregates might differ between those formed by simple salts and matrix‐forming heparin. We propose that a conformational phase diagram, accommodating crystal‐like amyloid fibrils and glass‐like amorphous aggregates, is important for understanding the effects of various additives.     

Preparation of (2S,4R)‐2,4‐thiazolidinedicarboxylic acid by separation of diastereoisomeric salts with organic amines

L ‐Cysteine was condensed with glyoxylic acid monohydrate in acetic acid at 30°C to give (4R)‐2,4‐thiazolidinedicarboxylic acid [(4R)‐TDA] as a mixture of two diastereoisomers, (2R,4R)‐ and (2S,4R)‐TDA. An attempt was made to separate (2S,4R)‐TDA from the diastereoisomeric salts of (4R)‐TDA with 1‐propylamine, 2‐methyl‐2‐propylamine, benzylamine, and (R)‐ and (S)‐1‐phenylethylamines [(R)‐ and (S)‐PEA]. The salts of (2S,4R)‐TDA were preferentially crystallized as less soluble diastereoisomeric salts. When the salt with (R)‐PEA was employed, the separation was successfully achieved to afford optically pure (2S,4R)‐TDA in a yield of 41%, based on the starting amount of the diastereoisomeric mixture of (4R)‐TDA. Chirality 11:326–329, 1999. © 1999 Wiley‐Liss, Inc.     

Crystal structures of carbohydrate recognition domain of blood dendritic cell antigen‐2 (BDCA2) reveal a common domain‐swapped dimer

We report on crystal structures of a carbohydrate recognition domain (CRD) of human C‐type lectin receptor blood dendritic cell antigen‐2 (BDCA2). Three different crystal forms were obtained at 1.8–2.3 Å resolution. In all three, the CRD has a basic C‐type lectin fold, but a long loop extends away from the core domain to form a domain‐swapped dimer. The structures of the dimers from the three different crystal forms superimpose well, indicating that domain swapping and dimer formation are energetically stable. The structure of the dimer is compared with other domain‐swapped proteins, and a possible regulation mechanism of BDCA2 is discussed. Proteins 2014; 82:1512–1518. © 2013 Wiley Periodicals, Inc.