Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity

Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major variant surface antigen, variant surface glycoprotein (VSG), to evade the host immune response, and VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double-strand breaks (DSBs) in both active and silent subtelomeric bloodstream form expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion induced much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES.     

Pentose phosphate metabolism during dormancy breakage in Corylus avellana L.

Commercially obtained fruits of Corylus avellana exhibit the characteristic loss of dormancy of this seed following chilling under moist conditions. The activities of cytosolic and organellar enzymes of pentose phosphate pathway in cotyledonary tissue were assayed throughout stratification and over a similar period in damp vermiculite at 20° C. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconic acid dehydrogenase (6PGDH) were both found in cytosolic extracts in all treatments; only 6PGDH was present in the organellar fraction.The enzyme activities monitored in seeds at 20° C remained relatively constant over the course of the investigation except in the case of cytosolic 6PGDH where it is suggested an inhibitor of the enzyme accumulated. This inhibitor was removed by the partial purification procedure. Increases in the activities of the enzymes occurred during stratification, the major increase coinciding exactly with dormancy breakage but prior to the initiation of germination. The marked increase in G6PDH and 6PGDH concurrent with the change in germination potential of the chilled seed may have considerable biochemical significance in breaking down the dormant state.Abbreviations G6P glucose-6-phosphate - G6PDH glucose-6 phosphate dehydrogenase - NADP nicotinamide adenine dinucleotide phosphate - 6 PGDH 6-phosphogluconic acid dehydrogenase - PPP pentose phosphate pathway     

The Three-dimensional Structure of a Mycobacterial DapD Provides Insights into DapD Diversity and Reveals Unexpected Particulars about the Enzymatic Mechanism

The enzyme tetrahydrodipicolinate N-succinyltransferase (DapD) is part of the L-lysine biosynthetic pathway. This pathway is crucial for the survival of the pathogen Mycobacterium tuberculosis (Mtb) and, consequently, the enzymes of the pathway are potential drug targets. We report here the crystal structures of Mtb-DapD and of Mtb-DapD in complex with the co-factor succinyl-CoA (SCoA) at 2.15 Å and 1.97 Å resolution, respectively. Each subunit of the trimeric enzyme consists of three domains, of which the second, a left-handed, parallel β-helix (LβH domain), is the common structural motif of enzymes belonging to the hexapeptide repeat superfamily. The trimeric quaternary structure is stabilized by Mg²⁺ and Na⁺ located on the 3-fold axis. The binary complex of Mtb-DapD and SCoA reveals the binding mode(s) of the co-factor and a possible covalent reaction intermediate. The N-terminal domain of Mtb-DapD exhibits a unique architecture, including an interior water-filled channel, which allows access to a magnesium ion located at the 3-fold symmetry axis.     

Ordering of C-terminal loop and glutaminase domains of glucosamine-6-phosphate synthase promotes sugar ring opening and formation of the ammonia channel

Glucosamine-6-phosphate synthase (GlmS) channels ammonia from glutamine at the glutaminase site to fructose 6-phosphate (Fru6P) at the synthase site. Escherichia coli GlmS is composed of two C-terminal synthase domains that form the dimer interface and two N-terminal glutaminase domains at its periphery. We report the crystal structures of GlmS alone and in complex with the glucosamine-6-phosphate product at 2.95 Å and 2.9 Å resolution, respectively. Surprisingly, although the whole protein is present in this crystal form, no electron density for the glutaminase domain was observed, indicating its mobility. Comparison of the two structures with that of the previously reported GlmS-Fru6P complex shows that, upon sugar binding, the C-terminal loop, which forms the major part of the channel walls, becomes ordered and covers the synthase site. The ordering of the glutaminase domains likely follows Fru6P binding by the anchoring of Trp74, which acts as the gate of the channel, on the closed C-terminal loop. This is accompanied by a major conformational change of the side chain of Lys503^# of the neighboring synthase domain that strengthens the interactions of the synthase domain with the C-terminal loop and completely shields the synthase site. The concomitant conformational change of the Lys503^#-Gly505^# tripeptide places catalytic His504^# in the proper position to open the sugar and buries the linear sugar, which is now in the vicinity of the catalytic groups involved in the sugar isomerization reaction. Together with the previously reported structures of GlmS in complex with Fru6P or glucose 6-phosphate and a glutamine analogue, the new structures reveal the structural changes occurring during the whole catalytic cycle.     

Structural Insights into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2₁ at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.     

Molecular Cloning and Crystal Structural Analysis of a Novel β-N-Acetylhexosaminidase from Paenibacillus sp. TS12 Capable of Degrading Glycosphingolipids

We report the molecular cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 Å; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 Å; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 Å. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids.     

Conformational Changes in a Plant Ketol-Acid Reductoisomerase upon Mg and NADPH Binding as Revealed by Two Crystal Structures

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) is an enzyme in the branched-chain amino acid biosynthesis pathway where it catalyzes the conversion of 2-acetolactate into (2R)-2,3-dihydroxy-3-isovalerate or the conversion of 2-aceto-2-hydroxybutyrate into (2R,3R)-2,3-dihydroxy-3-methylvalerate. KARI catalyzes two reactions—alkyl migration and reduction—and requires Mg²⁺ and NADPH for activity. To date, the only reported structures for a plant KARI are those of the spinach enzyme-Mn²⁺-(phospho)ADP ribose-(2R,3R)-2,3-dihydroxy-3-methylvalerate complex and the spinach KARI-Mg²⁺-NADPH-N-hydroxy-N-isopropyloxamate complex, where N-hydroxy-N-isopropyloxamate is a predicted transition-state analog. These studies demonstrated that the enzyme consists of two domains, N-domain and C-domain, with the active site at the interface of these domains. Here, we have determined the structures of the rice KARI-Mg²⁺ and rice KARI-Mg²⁺-NADPH complexes to 1.55 Å and 2.80 Å resolutions, respectively. In comparing the structures of all the complexes, several differences are observed. Firstly, the N-domain is rotated up to 15° relative to the C-domain, expanding the active site by up to 4 Å. Secondly, an α-helix in the C-domain that includes residues V510-T519 and forms part of the active site moves by ∼ 3.9 Å upon binding of NADPH. Thirdly, the 15 C-terminal amino acid residues in the rice KARI-Mg²⁺ complex are disordered. In the rice KARI-Mg²⁺-NADPH complex and the spinach KARI structures, many of the 15 residues bind to NADPH and the N-domain and cover the active site. Fourthly, the location of the metal ions within the active site can vary by up to 2.7 Å. The new structures allow us to propose that an induced-fit mechanism operates to (i) allow substrate to enter the active site, (ii) close over the active site during catalysis, and (iii) open the active site to facilitate product release.     

The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P2₁. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg²⁺ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.     

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of α-1,6 glucosidic linkages in limit dextrins. This activity plays a role in starch degradation during germination and presumably in starch biosynthesis during grain filling. The crystal structures of HvLD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight-stranded β-sandwich domain, a six-stranded β-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (β/α)₈-like barrel domain that lacks α-helix 5, and a C-terminal eight-stranded β-sandwich domain of unknown function. The CDs are bound at the active site occupying carbohydrate binding subsites + 1 and + 2. A glycerol and three water molecules mimic a glucose residue at subsite − 1, thereby identifying residues involved in catalysis. The bulky Met440, a unique residue at its position among α-1,6 acting enzymes, obstructs subsite − 4. The steric hindrance observed is proposed to affect substrate specificity and to cause a low activity of HvLD towards amylopectin. An extended loop (Asp513-Asn520) between β5 and β6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for α-CD than pullulanases. The crystal structures additionally provide new insight into cation sites and the concerted action of the battery of hydrolytic enzymes in starch degradation.     

Structural basis for natural lactonase and promiscuous phosphotriesterase activities

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 Å resolution) and in complex with a quorum-sensing lactone mimic at 2.0 Å resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

Crystal Structures of A. acidocaldarius Endoglucanase Cel9A in Complex with Cello-Oligosaccharides: Strong − 1 and − 2 Subsites Mimic Cellobiohydrolase Activity

Alicyclobacillus acidocaldarius endoglucanase Cel9A (AaCel9A) is an inverting glycoside hydrolase with β-1,4-glucanase activity on soluble polymeric substrates. Here, we report three X-ray structures of AaCel9A: a ligand-free structure at 1.8 Å resolution and two complexes at 2.66 and 2.1 Å resolution, respectively, with cellobiose obtained by co-crystallization and with cellotetraose obtained by the soaking method. AaCel9A forms an (α/α)₆-barrel like other glycoside hydrolase family 9 enzymes. When cellobiose is used as a ligand, three glucosyl binding subsites are occupied, including two on the glycone side, while with cellotetraose as a ligand, five subsites, including four on the glycone side, are occupied. A structural comparison showed no conformational rearrangement of AaCel9A upon ligand binding. The structural analysis demonstrates that of the four minus subsites identified, subsites − 1 and − 2 show the strongest interaction with bound glucose. In conjunction with the open active-site cleft of AaCel9A, this is able to reconcile the previously observed cleavage of short-chain oligosaccharides with cellobiose as main product with the endo mode of action on larger polysaccharides.     

The Crystal Structure of ATP-bound Phosphofructokinase from Trypanosoma brucei Reveals Conformational Transitions Different from those of Other Phosphofructokinases

The crystal structure of the ATP-bound form of the tetrameric phosphofructokinase (PFK) from Trypanosoma brucei enables detailed comparisons to be made with the structures of the apoenzyme form of the same enzyme, as well as with those of bacterial ATP-dependent and PP_i-dependent PFKs. The active site of T. brucei PFK (which is strictly ATP-dependent but belongs to the PP_i-dependent family by sequence similarities) is a chimera of the two types of PFK. In particular, the active site of T. brucei PFK possesses amino acid residues and structural features characteristic of both types of PFK. Conformational changes upon ATP binding are observed that include the opening of the active site to accommodate the two substrates, MgATP and fructose 6-phosphate, and a dramatic ordering of the C-terminal helices, which act like reaching arms to hold the tetramer together. These conformational transitions are fundamentally different from those of other ATP-dependent PFKs. The substantial differences in structure and mechanism of T. brucei PFK compared with bacterial and mammalian PFKs give optimism for the discovery of species-specific drugs for the treatment of diseases caused by protist parasites of the trypanosomatid family.     

Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 °C and withstanding incubation at temperatures up to 70 °C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 Å. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme.     

A kinetoplastid BRCA2 interacts with DNA replication protein CDC45

The gene BRCA2, first identified as a breast cancer susceptibility locus in humans, encodes a protein involved in DNA repair in mammalian cells and mutations in this gene confer increased risk of breast cancer. Here we report a functional characterisation of a Trypanosoma brucei BRCA2 (TbBRCA2) orthologue and show that the protein interacts directly with TbRAD51. A further protein-protein interaction screen using TbBRCA2 identified other interacting proteins, including a trypanosome orthologue of CDC45 which is involved in initiation and progression of the replication fork complex during DNA synthesis. Deletion of the TbBRCA2 gene retards cell cycle progression during S-phase as judged by increased incorporation of BrdU and an increased percentage of cells with one nucleus and two kinetoplasts. These results provide insights into the potential role played by BRCA2 in DNA replication and reveal a novel interaction that couples replication and recombination in maintaining integrity of the genome.     

Expression and purification of non-glycosylated Trypanosoma brucei transferrin receptor in insect cells

The transferrin receptor of the parasite Trypanosoma brucei is a heterodimeric protein complex encoded by the 2 expression site-associated genes (ESAGs) 6 and 7. ESAG6 is a heterogeneously glycosylated protein of 50-60 kDa modified by a glycosylphosphatidylinositol anchor at the C-terminus, while ESAG7 is a 40-42 kDa glycoprotein carrying an unmodified C-terminus. In order to determine whether glycosylation is necessary for dimer formation and ligand binding, the receptor was expressed in insect cells in the presence of tunicamycin. When insect cells were infected with recombinant ESAG6/ESAG7 double expressor baculovirus and grown in the presence of tunicamycin, non-glycosylated forms of ESAG6 and ESAG7 of 46 and 36 kDa, respectively, were synthesized. The non-glycosylated ESAG6 and ESAG7 were capable of forming a heterodimer and of binding transferrin. This results shows that glycosylation is not necessary for synthesis of a functional T. brucei transferrin receptor.     

The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity

Pseudomonas stutzeril-rhamnose isomerase (P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli (E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with l-rhamnose and d-allose which has different configurations of C4 and C5 from l-rhamnose, were determined at a resolution of 2.0 Å, 1.97 Å, and 1.97 Å, respectively. P. stutzeri L-RhI has a large domain with a (β/α)₈ barrel fold and an additional small domain composed of seven α-helices, forming a homo tetramer, as found in E. coli L-RhI and d-xylose isomerases (D-XIs) from various microorganisms. The β1-α1 loop (Gly60-Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding β1-α1 loop is extended (Asp52-Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with l-rhamnose and d-allose show that both substrates are nicely fitted to the substrate -binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the β1-α1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of l-rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.     

The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation

Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3′-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein, TbNCP1. RNAi targeting PUF7 reduced trypanosome growth and inhibited two steps in ribosomal RNA processing.     

Crystal Structure Analysis of Free and Substrate-Bound 6-Hydroxy-l-Nicotine Oxidase from Arthrobacter nicotinovorans

The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-l-nicotine oxidase (6HLNO) and 6-hydroxy-d-nicotine oxidase, which act with absolute stereospecificity on the l- and d-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 Å resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-l-nicotine at 2.05 Å resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-d-nicotine oxidase, based on models of complexes with the d-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.