Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme.

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) from Staphylococcus lugdunensis.

The 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) catalyzes final two steps of purine nucleotide de novo biosynthetic pathway. This study reports the characterization of ATIC from Staphylococcus lugdunensis (SlugATIC). Apart from kinetic analysis and a detailed biophysical characterization of SlugATIC, the role of ATIC in cell proliferation has been demonstrated for the first time. The purified recombinant SlugATIC and its truncated domains exist mainly in dimeric form was revealed in gel-filtration and glutaraldehyde cross-linking studies. The two activities reside on separate domains was demonstrated in kinetic analysis of SlugATIC and reconstituted truncated N-terminal IMP cyclohydrolase (IMPCHase) and C-terminal AICAR transformylase (AICAR TFase) domains. Site-directed mutagenesis showed that Lys255 and His256 are the key catalytic residues, while Asn415 substantially contributes to AICAR TFase activity in SlugATIC. The differential scanning calorimetry (DSC) analysis revealed a molten globule-like structure for independent N-terminal domain as compared with a relatively stable conformational state in full-length SlugATIC signifying the importance of covalently linked domains. Unlike reported crystal structures, the DSC studies revealed significant conformational changes on binding of leading ligand to AICAR TFase domain in SlugATIC. The cell proliferation activity of SlugATIC was observed where it promoted proliferation and viability of NIH 3T3 and RIN-5F cells, exhibited in vitro wound healing in NIH 3T3 fibroblast cells, and rescued RIN-5F cells from the cytotoxic effects of palmitic acid and high glucose. The results suggest that ATIC, an important drug target, can also be exploited for its cell proliferative properties.

A genome-wide association study reveals a novel candidate gene for sperm motility in pigs.

Sperm motility is one of the most widely used parameters in order to evaluate boar semen quality. However, this trait can only be measured after puberty. Thus, the use of genomic information appears as an appealing alternative to evaluate and improve selection for boar fertility traits earlier in life. With this study we aimed to identify SNPs with significant association with sperm motility in two different commercial pig populations and to identify possible candidate genes within the identified QTL regions. We performed a single-SNP genome-wide association study using genotyped animals from a Landrace-based (L1) and a Large White-based (L2) pig populations. For L1, a total of 602 animals genotyped for 42,551 SNPs were used in the association analysis. For L2, a total of 525 animals genotyped for 40,890 SNPs were available. After the association analysis, a false discovery rate q-value ≤0.05 was used as the threshold for significant association. No SNPs were significantly associated with sperm motility in L1, while six SNPs on Sus scrofa chromosome 1 (position 117.26-119.56Mb) were significant in L2. The mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene, which affects translation efficiency of proteins in sperm cells, was identified as a putative candidate gene. The significant markers identified in this study may be useful to enhance the genetic improvement of sperm motility by selection of boars at an earlier age under a marker assisted selection strategy.

A novel function for the N-terminal nucleophile hydrolase fold demonstrated by the structure of an archaeal inosine monophosphate cyclohydrolase.

Inosine 5'-monophosphate (IMP) cyclohydrolase catalyzes the cyclization of 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) to IMP in the final step of de novo purine biosynthesis. Two major types of this enzyme have been discovered to date: PurH in Bacteria and Eukarya and PurO in Archaea. The structure of the MTH1020 gene product from Methanothermobacter thermoautotrophicus was previously solved without functional annotation but shows high amino acid sequence similarity to other PurOs. We determined the crystal structure of the MTH1020 gene product in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) at 2.0 and 2.6 A resolution, respectively. On the basis of the sequence analysis, ligand-bound structures, and biochemical data, MTH1020 is confirmed as an archaeal IMP cyclohydrolase, thus designated as MthPurO. MthPurO has a four-layered alphabeta betaalpha core structure, showing an N-terminal nucleophile (NTN) hydrolase fold. The active site is located at the deep pocket between two central beta-sheets and contains residues strictly conserved within PurOs. Comparisons of the two types of IMP cyclohydrolase, PurO and PurH, revealed that there are no similarities in sequence, structure, or the active site architecture, suggesting that they are evolutionarily not related to each other. The MjR31K mutant of PurO from Methanocaldococcus jannaschii showed 76% decreased activity and the MjE102Q mutation completely abolished enzymatic activity, suggesting that these highly conserved residues play critical roles in catalysis. Interestingly, green fluorescent protein (GFP), which has no structural homology to either PurO or PurH but catalyzes a similar intramolecular cyclohydrolase reaction required for chromophore maturation, utilizes Arg96 and Glu222 in a mechanism analogous to that of PurO.

Modular organization of FDH: Exploring the basis of hydrolase catalysis.

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.

Purification, crystallization and preliminary crystallographic analysis of the glycine-cleavage system component T-protein from Pyrococcus horikoshii OT3.

The glycine-cleavage system component T-protein is a folate-dependent enzyme that catalyzes the formation of ammonia and 5,10-CH2-tetrahydrofolate from the aminomethyl intermediate bound to the lipoate cofactor of H-protein. T-protein from Pyrococcus horikoshii OT3 has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. X-ray diffraction data have been collected to 1.50 A resolution at 100 K using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 78.980, b = 95.708, c = 118.331 A. Assuming one homodimer per asymmetric unit gives a VM value of 2.4 A3 Da(-1) and a solvent content of 49.0%.

Mycobacterium tuberculosis ketopantoate hydroxymethyltransferase: tetrahydrofolate-independent hydroxymethyltransferase and enolization reactions with alpha-keto acids.

The panB gene that encodes ketopantoate hydroxymethyltransferase has been cloned from Mycobacterium tuberculosis, expressed, and purified to homogeneity. 1H NMR spectroscopy was used to determine the rate of (i) tetrahydrofolate-independent hydroxymethyltransferase chemistry between formaldehyde and alpha-ketoisovalerate and (ii) deuterium exchange in the methylenetetrahydrofolate-independent enolization of alpha-ketoisovalerate and other alpha-keto acids, catalyzed by PanB. These studies have demonstrated that substrate enolization by PanB is divalent metal-dependent with a preference of Mg2+ > Zn2+ > Co2+ > Ni2+ > Ca2+. The rate of enolization is pH-dependent with optimal activity in the range of 7.0-7.5. The pH profile was bell-shaped, depending on the ionization state of two ionizable groups with apparent pK values of 6.2 and 8.3. Enolization and isotope exchange occurs with some alpha-keto acids (e.g., pyruvate and alpha-ketobutyrate), resulting in the complete exchange of all beta-hydrogens. Enzyme-catalyzed enolization and isotope exchange occur with other long-chain and branched alpha-keto acids, resulting in the stereospecific exchange of only one of the beta-hydrogen atoms. These results are discussed in the context of steric restrictions present in the enzyme active site and the stereochemistry of base-catalyzed isotope exchange.

Structural characterization of T-protein of the Escherichia coli glycine cleavage system by X-ray small angle scattering.

T-protein, one of the components of the glycine cleavage complex, catalyses the formation of ammonia and methylene-tetrahydrofolate from H-protein-bound intermediate. Native T-protein of the glycine cleavage system from E. coli was efficiently purified using a combination of hydrophobic interaction, gel permeation and ion exchange chromatography. Synchrotron radiation small angle X-ray solution scattering indicates that T-protein has an extended structure in solution. A low resolution model of the protein was constructed ab initio and tentative models of the tertiary structure were built using prediction methods constrained by the scattering data.

The kinetic mechanism of the human bifunctional enzyme ATIC (5-amino-4-imidazolecarboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase). A surprising lack of substrate channeling.

5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.

Characterization of the formyltransferase from Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 possesses a formaldehyde-oxidation pathway that involves enzymes with high sequence identity with enzymes from methanogenic and sulfate-reducing archaea. Here we describe the purification and characterization of formylmethanofuran-tetrahydromethanopterin formyltransferase (Ftr), which catalyzes the reversible formation of formylmethanofuran (formylMFR) and tetrahydromethanopterin (H4MPT) from N5-formylH4MPT and methanofuran (MFR). Formyltransferase from M. extorquens AM1 showed activity with MFR and H4MPT isolated from the methanogenic archaeon Methanothermobacter marburgensis (apparent Km for formylMFR = 50 microM; apparent Km for H4MPT = 30 microM). The enzyme is encoded by the ffsA gene and exhibits a sequence identity of approximately 40% with Ftr from methanogenic and sulfate-reducing archaea. The 32-kDa Ftr protein from M. extorquens AM1 copurified in a complex with three other polypeptides of 60 kDa, 37 kDa and 29 kDa. Interestingly, these are encoded by the genes orf1, orf2 and orf3 which show sequence identity with the formylMFR dehydrogenase subunits FmdA, FmdB and FmdC, respectively. The clustering of the genes orf2, orf1, ffsA, and orf3 in the chromosome of M. extorquens AM1 indicates that, in the bacterium, the respective polypeptides form a functional unit. Expression studies in Escherichia coli indicate that Ftr requires the other subunits of the complex for stability. Despite the fact that three of the polypeptides of the complex showed sequence similarity to subunits of Fmd from methanogens, the complex was not found to catalyze the oxidation of formylMFR. Detailed comparison of the primary structure revealed that Orf2, the homolog of the active site harboring subunit FmdB, lacks the binding motifs for the active-site cofactors molybdenum, molybdopterin and a [4Fe-4S] cluster. Cytochrome c was found to be spontaneously reduced by H4MPT. On the basis of this property, a novel assay for Ftr activity and MFR is described.

Crystallization and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4.

Deoxycytidylate hydroxymethylase from bacteriophage T4 is a homodimeric enzyme in which each polypeptide chain consists of 246 amino-acid residues. It has been crystallized in the presence of its substrate, deoxycytidine monophosphate, at room temperature using sodium citrate as precipitant. The crystals are monoclinic, belonging to space group C2, with unit-cell parameters a = 174.22, b = 53.12, c = 75.17 A, beta = 115.29 degrees. The asymmetric unit contains one homodimer, with a corresponding Vm of 2.65 A3 Da-1 and solvent content of 54%. Native diffraction data to 1.6 A resolution have been collected from two crystals using synchrotron radiation.

Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNAMet(f) formyltransferase complexed with formyl-methionyl-tRNAMet(f).

The structure of methionyl-tRNAfMet(f) formyltransferase from E. coli, a monomeric protein of 34 kDa, has previously been determined at 2.0 A resolution. In the present work, this enzyme was crystallized as a complex with its macromolecular product, the initiator formyl-methionyl-tRNAfMet(f) (25 kDa). Polyethylene glycol 5000 monomethylether was used as a precipitating agent. The crystals are orthorhombic and have unit-cell parameters a = 201.7, b = 68.1, c = 86.4 A. They belong to space group P21212 and diffract to 2.8 A resolution. The structure is being solved with the help of a mercury derivative.

Expression and characterization of bovine mitochondrial methionyl-tRNA transformylase.

Translational initiation in bacteria and some organelles such as mitochondria and chloroplasts requires formyl-methionyl-tRNA (fMet-tRNA). Methionyl-tRNA (Met-tRNA) undergoes formylation by methionyl-tRNA transformylase (MTF), and the resulting fMet-tRNA is utilized exclusively in the initiation process. The gene encoding mammalian mitochondrial MTF (MTFmt) was cloned recently. When the cDNA corresponding to mature MTFmt was cloned into an expression vector, no expression of MTFmt was observed. However, if the cDNA was fused with the histidine-tag sequence at the N-terminus, MTFmt could be expressed in Escherichia coli. The recombinant enzyme was purified by a single step on a histidine-binding metal affinity column. We previously found that native MTFmt is able to formylate E. coli elongator Met-tRNA as well as the initiator Met-tRNA. The specific formylation of the initiator Met-tRNA by E. coli MTF is quite important in bacterial translational initiation. The purified recombinant MTFmt with the histidine-tag showed almost identical kinetic parameters to those of native MTFmt. This expression system is suitable for the rapid, efficient production of MTFmt in amounts adequate for further biophysical studies, which will provide another approach for elucidating the formylation mechanism, in addition to studies on E. coli MTF.

C1 transfer enzymes and coenzymes linking methylotrophic bacteria and methanogenic Archaea.

Methanogenic and sulfate-reducing Archaea are considered to have an energy metabolism involving C1 transfer coenzymes and enzymes unique for this group of strictly anaerobic microorganisms. An aerobic methylotrophic bacterium, Methylobacterium extorquens AM1, was found to contain a cluster of genes that are predicted to encode some of these enzymes and was shown to contain two of the enzyme activities and one of the methanogenic coenzymes. Insertion mutants were all unable to grow on C1 compounds, suggesting that the archaeal enzymes function in aerobic C1 metabolism. Thus, methylotrophy and methanogenesis involve common genes that cross the bacterial/archaeal boundaries.

The human trifunctional enzyme of de novo purine biosynthesis: heterologous expression, purification, and preliminary characterization.

The cDNA for the human trifunctional enzyme of de novo purine biosynthesis, which encodes glycinamide ribonucleotide synthetase, aminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide trans-formylase, has been overexpressed in Escherichia coli and its protein product has been purified to homogeneity. The glycinamide ribonucleotide transformylase activity, which constitutes the C-terminal domain of the trifunctional enzyme, has been characterized with respect to its kinetic constants, Vmax = 3.03 +/- 0.15 micromol/min-mg and Km values for beta-glycinamide ribonucleotide and 10-formyl-5,8-dideazafolate of 0.94 +/- 0.21 and 1.58 +/- 0.25 microM, respectively, and its kinetic mechanism, which is ordered-sequential with the folate substrate binding first. The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.

Characterization of molecularly cloned human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.

The cDNA encoding human 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase has been cloned from a placenta cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 592 amino acids. The amino (N)-terminal sequence of this enzyme, purified from HeLa cells and CCRF-CEM cells, was found to be APGQLALF-. Both sequencing results revealed a difference of six N-terminal residues when compared to the reported sequence of cloned cDNA from a hepatoma cDNA library. Northern-blot analysis of human AICAR transformylase mRNA showed the expression of a single 2.0 kb mRNA in all tissues examined. With the cloned cDNA fragment, we constructed expression vectors for mature and GST-fused AICAR transformylase. Both recombinant molecules possessing AICAR transformylase activity were overproduced in Escherichia coli. GST-AICAR transformylase can be purified to homogeneity by a single-step affinity procedure with glutathione Sepharose. Mutational analysis, utilizing this expression system, showed that His213 and His267 were essential for AICAR transformylase activity.