Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis

With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.     

The structure of glucose‐1‐phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA‐type enzymes

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six‐month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure‐based drug design is the enzyme RmlA, a glucose‐1‐phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l ‐rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here, we report the X‐ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP‐glucose to 1.6 Å and 1.85 Å resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP‐glucose complex, only a single magnesium ion was observed. Importantly, for RmlA‐type enzymes with known three‐dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide‐linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure‐based drug design. In the past, molecular modeling procedures have been employed to derive a three‐dimensional model of the M. tuberculosis RmlA for drug design. The X‐ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases.     

Crystal structure of Saccharomyces cerevisiae Aro8, a putative α‐aminoadipate aminotransferase

α‐Aminoadipate aminotransferase (AAA‐AT) catalyzes the amination of 2‐oxoadipate to α‐aminoadipate in the fourth step of the α‐aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA‐AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2‐oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA‐AT LysN from Thermus thermophilus and human kynurenine aminotransferase II. Inspection of the active site of Aro8 reveals asymmetric cofactor binding with lysine‐pyridoxal‐5‐phosphate bound within the active site of one subunit in the Aro8 homodimer and pyridoxamine phosphate and a HEPES molecule bound to the other subunit. The HEPES buffer molecule binds within the substrate‐binding site of Aro8, yielding insights into the mechanism by which it recognizes multiple substrates and how this recognition differs from other AAA‐AT/kynurenine aminotransferases.     

Structure and properties of recombinant human pyridoxine 5'-phosphate oxidase

Pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5'-phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of approximately 0.2 sec(-1) and K(m) values in the low micromolar range for both pyridoxine 5'phosphate and pyridoxamine 5'-phosphate. Pyridoxal 5'-phosphate is an effective product inhibitor. The three-dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5'-phosphate tightly on each subunit.     

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.     

Conformational transitions driven by pyridoxal‐5′‐phosphate uptake in the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii

Serine hydroxymethyltransferase (SHMT) is a pyridoxal‐5′‐phosphate (PLP)‐dependent enzyme belonging to the fold type I superfamily, which catalyzes in vivo the reversible conversion of l ‐serine and tetrahydropteroylglutamate (H₄PteGlu) to glycine and 5,10‐methylenetetrahydropteroylglutamate (5,10‐CH₂‐H₄PteGlu). The SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) had been recently purified and characterized. This enzyme was shown to display catalytic and stability properties typical of psychrophilic enzymes, namely high catalytic activity at low temperature and thermolability. To gain deeper insights into the structure–function relationship of piSHMT, the three‐dimensional structure of its apo form was determined by X‐ray crystallography. Homology modeling techniques were applied to build a model of the piSHMT holo form. Comparison of the two forms unraveled the conformation modifications that take place when the apo enzyme binds its cofactor. Our results show that the apo form is in an “open” conformation and possesses four (or five, in chain A) disordered loops whose electron density is not visible by X‐ray crystallography. These loops contain residues that interact with the PLP cofactor and three of them are localized in the major domain that, along with the small domain, constitutes the single subunit of the SHMT homodimer. Cofactor binding triggers a rearrangement of the small domain that moves toward the large domain and screens the PLP binding site at the solvent side. Comparison to the mesophilic apo SHMT from Salmonella typhimurium suggests that the backbone conformational changes are wider in psychrophilic SHMT. Proteins 2014; 82:2831–2841. © 2014 Wiley Periodicals, Inc.     

Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase.

Pyridoxine-5'-phosphate oxidase catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to an aldehyde, forming pyridoxal 5'-phosphate. A hydrogen atom is removed from C4' during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5'-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle. Using (4'R)-[(3)H]PMP as substrate, nearly 100 % of the radiolabel appears in water after oxidation to pyridoxal 5'-phosphate. Thus, the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon atom of pyridoxamine 5'-phosphate. Site mutants were made of all residues at the active site that interact with the oxygen atom or amine group on C4' of the substrates. Other residues that make interactions with the phosphate moiety of the substrate were mutated. The mutants showed a decrease in affinity, but exhibited considerable catalytic activity, showing that these residues are important for binding, but play a lesser role in catalysis. The exception is Arg197, which is important for both binding and catalysis. The R197 M mutant enzyme catalyzed removal of the proS hydrogen atom from (4'R)-[(3)H]PMP, showing that the guanidinium side-chain plays an important role in determining stereospecificity. The crystal structure and the stereospecificity studies suggests that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.     

Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti

Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose–methanol–choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His₆-tag and PNOX–pyridoxamine (PM) complex were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His–Asn or His–His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.     

The crystal structure of the mycobacterium tuberculosis Rv3019c-Rv3020c ESX complex reveals a domain-swapped heterotetramer

Mycobacterium tuberculosis encodes five gene clusters (ESX‐1 to ESX‐5) for Type VII protein secretion systems that are implicated in mycobacterial pathogenicity. Substrates for the secretion apparatus are encoded within the gene clusters and in additional loci that lack the components of the secretion apparatus. The best characterized substrates are the ESX complexes, 1:1 heterodimers of ESAT‐6 and CFP‐10, the prototypical member that has been shown to be essential for Mycobacterium tuberculosis pathogenesis. We have determined the structure of EsxRS, a homolog of EsxGH of the ESX‐3 gene cluster, at 1.91 Å resolution. The EsxRS structure is composed of two four‐helix bundles resulting from the 3D domain swapping of the C‐terminal domain of EsxS, the CFP‐10 homolog. The four‐helix bundles at the extremities of the complex have a similar architecture to the structure of ESAT‐6·CFP‐10 (EsxAB) of ESX‐1, but in EsxRS a hinge loop linking the α‐helical domains of EsxS undergoes a loop‐to‐helix transition that creates the domain swapped EsxRS tetramer. Based on the atomic structure of EsxRS and existing biochemical data on ESX complexes, we propose that higher order ESX oligomers may increase avidity of ESX binding to host receptor molecules or, alternatively, the conformational change that creates the domain swapped structure may be the basis of ESX complex dissociation that would free ESAT‐6 to exert a cytotoxic effect.     

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

Crystal structure of calcium dodecin (Rv0379), from Mycobacterium tuberculosis with a unique calcium-binding site

In eukaryotes, calcium-binding proteins play a pivotal role in diverse cellular processes, and recent findings suggest similar roles for bacterial proteins at different stages in their life cycle. Here, we report the crystal structure of calcium dodecin, Rv0379, from Mycobacterium tuberculosis with a dodecameric oligomeric assembly and a unique calcium-binding motif. Structure and sequence analysis were used to identify orthologs of Rv0379 with different ligand-binding specificity.     

5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

The structure of a putative S‐formylglutathione hydrolase from Agrobacterium tumefaciens

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Å resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the α/β hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes C—O bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed.     

Crystal structure of the protein from Arabidopsis thaliana gene At5g06450, a putative DnaQ‐like exonuclease domain‐containing protein with homohexameric assembly

Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ‐like 3′‐5′ exonuclease domain‐containing protein (AtDECP). The DnaQ‐like 3′‐5′ exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed β‐sheet flanked by α‐helices. Interestingly, AtDECP forms a homohexameric assembly with a central six fold symmetry, generating a central cavity. The ring‐shaped structure and comparison with WRN‐exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N‐terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ‐like 3′‐5′ exonuclease and its substrate nucleic acids.Proteins 2013. © 2013 Wiley Periodicals, Inc.