Biochemical and structural studies of two tetrameric nucleoside 2'-deoxyribosyltransferases from psychrophilic and mesophilic bacteria: Insights into cold-adaptation.

Nucleoside 2'-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2'-deoxynucleosides and the following transfer of the 2'-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures.

Fragments as Novel Starting Points for tRNA-Guanine Transglycosylase Inhibitors Found by Alternative Screening Strategies.

Crystallography provides structural information crucial for fragment optimization, however several criteria must be met to screen directly on protein crystals as soakable, well-diffracting specimen must be available. We screened a 96-fragment library against the tRNA-modifying enzyme TGT using crystallography. Eight hits, some with surprising binding poses, were detected. However, the amount of data collection, reduction and refinement is assumed substantial. Therefore, having a reliable cascade of fast and cost-efficient methods available for pre-screening before embarking to elaborate crystallographic screening appears beneficial. This allows filtering of compounds to the most promising hits, available to rapidly progress from hit-to-lead. But how to ensure that this workflow is reliable? To answer this question, we also applied SPR and NMR to the same screening sample to study whether identical hits are retrieved. Upon hit-list comparisons, crystallography shows with NMR and SPR, only one overlapping hit and all three methods shared no common hits. This questions a cascade-type screening protocol at least in the current example. Compared to crystallography, SPR and NMR detected higher percentages of non-active-site binders suggesting the importance of running reporter ligand-based competitive screens in SPR and NMR, a requirement not needed in crystallography. Although not specific, NMR proved a more sensitive method relative to SPR and crystallography, as it picked up the highest numbers of binders.

Establishment of a high throughput-screening system for nucleoside deoxyribosyltransferase II mutant enzymes with altered substrate specificity.

Nucleoside deoxyribosyltransferase II (NDT) catalyzes the transglycosylation reaction of the 2'-deoxyribose moiety between purine and/or pyrimidine bases and has been widely used in the synthesis of nucleoside analogs. The high specificity of NDT for 2'-deoxyribose limits its applications. Because 2'C- and/or 3'C-modified nucleosides have been widely used as antiviral or antitumour agents, improving the activity of NDT towards these modified nucleosides by protein engineering is an area of interest to the pharmaceutical industry. NDT engineering is hindered by a lack of effective screening methods. This study developed a high-throughput screening system, which was established by nucleoside deoxyribosyltransferase II-cytidine deaminase co-expression, indophenol colorimetric assay and whole-cell catalysis. A high-throughput screening system for NDT was established for the first time. This system can be applied to detect NDT-specific activity for a variety of cytidine analogs with glycosyl and base modifications, such as 5-aza-2'-deoxycytidine, 2',3'-dideoxycytidine, cytosine-ß-d-arabinofuranoside. In this study, we adopted the semi-rational design of NDT and constructed a mutant library of NDT from Lactobacillus helveticus (LhNDT) by site-saturation mutagenesis. Over 600 mutants were screened, and a variant with up to a 5.2-fold higher conversion rate of 2',3'-dideoxyinosine was obtained.

Characterization of an atypical, thermostable, organic solvent- and acid-tolerant 2'-deoxyribosyltransferase from Chroococcidiopsis thermalis.

In our search for thermophilic and acid-tolerant nucleoside 2'-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical characterization revealed CtNDT as a homotetramer endowed with good activity and stability at both high temperatures (50-100 °C) and a wide range of pH values (from 3 to 7). CtNDT recognizes purine bases and their corresponding 2'-deoxynucleosides but is also proficient using cytosine and 2'-deoxycytidine as substrates. These unusual features preclude the strict classification of CtNDT as either a type I or a type II NDT and further suggest that this simple subdivision may need to be updated in the future. Our findings also hint at a possible link between oligomeric state and NDT's substrate specificity. Interestingly from a practical perspective, CtNDT displays high activity (80-100%) in the presence of several water-miscible co-solvents in a proportion of up to 20% and was successfully employed in the enzymatic production of several therapeutic nucleosides such as didanosine, vidarabine, and cytarabine.

Expression, purification and crystallization of phosphoribosyl transferase from a mycobacteriophage.

Tuberculosis (TB) continues to remain a leading cause of death globally. Of particular concern is the emergence and rise in incidence of multidrug-resistant and extremely drug-resistant cases of TB. To counter this threat, it is important to explore alternative therapies, including phage therapy. Phage BTCU-1 specifically infects Mycobacterium spp. and kills the majority of them. Intriguingly, many proteins from the phage do not share high amino-acid sequence identity with proteins from species other than phages. Here, the expression, purification and crystallization of one such protein, a putative phosphoribosyl transferase from phage BTCU-1, is reported. The crystals belonged to space group C2221, with unit-cell parameters a = 59.71, b = 64.42, c = 65.32 Å, α = ß = γ = 90°. The crystals diffracted X-rays to 2.2 Šresolution.

Identification of a Novel Mycobacterial Arabinosyltransferase Activity Which Adds an Arabinosyl Residue to α-d-Mannosyl Residues.

The arabinosyltransferases responsible for the biosynthesis of the arabinan domains of two abundant heteropolysaccharides of the cell envelope of all mycobacterial species, lipoarabinomannan and arabinogalactan, are validated drug targets. Using a cell envelope preparation from Mycobacterium smegmatis as the enzyme source and di- and trimannoside synthetic acceptors, we uncovered a previously undetected arabinosyltransferase activity. Thin layer chromatography, GC/MS, and LC/MS/MS analyses of the major enzymatic product are consistent with the transfer of an arabinose residue to the 6 position of the terminal mannosyl residue at the nonreducing end of the acceptors. The newly identified enzymatic activity is resistant to ethambutol and could correspond to the priming arabinosyl transfer reaction that occurs during lipoarabinomannan biosynthesis.

Composition, Assembly, and Trafficking of a Wheat Xylan Synthase Complex.

Xylans play an important role in plant cell wall integrity and have many industrial applications. Characterization of xylan synthase (XS) complexes responsible for the synthesis of these polymers is currently lacking. We recently purified XS activity from etiolated wheat (Triticum aestivum) seedlings. To further characterize this purified activity, we analyzed its protein composition and assembly. Proteomic analysis identified six main proteins: two glycosyltransferases (GTs) TaGT43-4 and TaGT47-13; two putative mutases (TaGT75-3 and TaGT75-4) and two non-GTs; a germin-like protein (TaGLP); and a vernalization related protein (TaVER2). Coexpression of TaGT43-4, TaGT47-13, TaGT75-3, and TaGT75-4 in Pichia pastoris confirmed that these proteins form a complex. Confocal microscopy showed that all these proteins interact in the endoplasmic reticulum (ER) but the complexes accumulate in Golgi, and TaGT43-4 acts as a scaffold protein that holds the other proteins. Furthermore, ER export of the complexes is dependent of the interaction between TaGT43-4 and TaGT47-13. Immunogold electron microscopy data support the conclusion that complex assembly occurs at specific areas of the ER before export to the Golgi. A di-Arg motif and a long sequence motif within the transmembrane domains were found conserved at the NH2-terminal ends of TaGT43-4 and homologous proteins from diverse taxa. These conserved motifs may control the forward trafficking of the complexes and their accumulation in the Golgi. Our findings indicate that xylan synthesis in grasses may involve a new regulatory mechanism linking complex assembly with forward trafficking and provide new insights that advance our understanding of xylan biosynthesis and regulation in plants.

Isolation and substrate specificity of an adenine nucleoside phosphorylase from adult Schistosoma mansoni.

An adenine nucleoside phosphorylase (ANP, EC none) activity was identified and partially purified from extracts of Schistosoma mansoni by chromatofocussing column chromatography and molecular sieving. The enzyme is distinct from purine nucleoside phosphorylase (PNP, EC 2.4.2.1). ANP is specific for adenine nucleosides which includes adenosine analogs modified in the aglycone, pentose or both moieties. (e.g. 2'-deoxyadenosine, 5'-deoxy-5'-methylthioadenosine, 5'-deoxy-5'-iodo-2-fluoroadenosine, etc.) The enzyme is also distinct from the mammalian 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP, EC 2.4.2.28) in that it is able of the phosphorolysis of 2'-deoxyadenosine while mammalian MTAP cannot. Because of ANP unique substrate specificity, the enzyme could play a role as a target for chemotherapy of these parasites. Cytotoxic analogs may be designed as subversive substrates that are selectively activated only by the schistosomal ANP.

Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli.

Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD+ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1 g L⁻¹ xylose and produced 15.5 g L⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⁻¹ xylose, 2.1 g L⁻¹ glucose and 1.5 g L⁻¹ arabinose, it produced a final succinate concentration of 17.2 g L⁻¹ with a yield of 0.94 g g⁻¹ total sugars.

In vitro assays of orphan glycosyltransferases and their application to identify Notch xylosyltransferases.

Here we describe a systematic approach to determine the activity of putative glycosyltransferases with a focus on orphan members of the glycosyltransferase 8 family. An assay that measures the hydrolysis activity of glycoslytransferases can indicate the donor nucleotide sugar specificity without previous knowledge about the acceptor. Knowing the donor specificity, the acceptor specificity can subsequently be determined using synthetic acceptors. Three putative glycosyltransferases, now renamed GXYLT1, GXYLT2, and XXYLT1, have been identified this way as xylosyltransferases and in addition have been shown to act on O-glucosylated EGF repeats of Notch.

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans.

Many archaeal tRNAs have archaeosine (G(+)) at position 15 in the D-loop and this is thought to strengthen the tertiary interaction with C48 in the V-loop. In the first step of G(+) biosynthesis, archaeosine tRNA-guanine transglycosylase (ArcTGT)(1) catalyzes the base exchange reaction from guanine to 7-cyano-7-deazaguanine (preQ(0)). ArcTGT is classified into full-size or split types, according to databases of genomic information. Although the full-size type forms a homodimeric structure, the split type has been assumed to form a heterotetrameric structure, consisting of two kinds of peptide. However, there has been no definitive evidence for this presented to date. Here, we show that native ArcTGT could be isolated from Methanosarcina acetivorans and two peptides formed a robust complex in cells. Consequently, the two peptides function as actual subunits of ArcTGT. We also overexpressed recombinant ArcTGT in Escherichia coli cells. Product was successfully obtained by co-overexpression of the two subunits but one subunit alone was not adequately expressed in soluble fractions. This result suggests that interaction between the two subunits may contribute to the conformational stability of split ArcTGT. The values of the kinetic parameters for the recombinant and native ArcTGT were closely similar. Moreover, tRNA transcript with preQ(0) at position 15 was successfully prepared using the recombinant ArcTGT. This tRNA transcript is expected to be useful as a substrate for studies seeking the enzymes responsible for G(+) biosynthesis.

Genotype MTBDRsl line probe assay shortens time to diagnosis of extensively drug-resistant tuberculosis in a high-throughput diagnostic laboratory.

RATIONALE: Conventional culture-based drug susceptibility testing (DST) for the second-line antituberculosis drugs is slow, leading to diagnostic delay with associated exacerbation of transmission, amplification of resistance, and increased mortality. OBJECTIVES: To assess the diagnostic performance of the GenoType MTBDRsl line probe assay (LPA) for the rapid detection of mutations conferring resistance to ofloxacin (OFX), amikacin (AMK), and ethambutol and to determine the impact of implementation on the turnaround time in a high-throughput diagnostic laboratory. METHODS: Six hundred and fifty-seven direct patient acid-fast bacilli smear-positive specimens resistant to isoniazid, rifampin, or both according to the GenoType MTBDRplus assay were consecutively tested, using the GenoType MTBDRsl LPA. The diagnostic performance was assessed relative to the "gold standard" culture-based method, and the laboratory turnaround times for both methods were determined. MEASUREMENTS AND MAIN RESULTS: A total of 516 of 657 patient specimens had valid results for both tests. The sensitivity for detecting OFX, AMK, and extensive drug resistance, using the GenoType MTBDRsl LPA, was 90.7% (95% confidence interval [CI], 80.1-96.0%), 100% (95% CI, 91.8-100%), and 92.3% (95% CI, 75.9-97.9%), respectively, and the specificity for detection was 98.1% (95% CI, 96.3-99.0%), 99.4% (95% CI, 98.2-99.8%), and 99.6% (95% CI, 98.5-99.9%), respectively. Implementation of this test significantly reduced the turnaround time by 93.3% (P < 0.001), calculated from the date that the specimen was received at the laboratory to reporting second-line results. In addition, a significant increase in diagnostic yield of 20.1% and 19.3% (P < 0.001) for OFX and AMK resistance, respectively, was obtained for isolates that were either contaminated or had lost viability. CONCLUSIONS: The GenoType MTBDRsl LPA is a rapid and reliable DST that can be easily incorporated into the diagnostic algorithm. This assay significantly improved diagnostic yield (P < 0.001) while simultaneously decreasing diagnostic delay for reporting second-line DST. The rapid dissemination of second-line DST results will guide initiation of appropriate treatment, thereby reducing transmission and improving treatment outcome.

Reconstitution of functional mycobacterial arabinosyltransferase AftC proteoliposome and assessment of decaprenylphosphorylarabinose analogues as arabinofuranosyl donors.

Arabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyltransferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac(2)PIM(2)), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC-liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose and linear α-d-Araf-(1→5)(3-5) oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced.

Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway.

RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2 days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.

First in-gel detection and purification of human xylosyltransferase II.

Human xylosyltransferases I and II (XylT-I, XylT-II) are key enzymes in glycosaminoglycan biosynthesis. Knowledge about the in vivo molecular weight, oligomeric state or turnover number are essential characteristics which have been addressed in this study. XylT-II was purified from Pichia pastoris by fractionated ammonium sulfate precipitation, heparin affinity and ion exchange chromatography. XylT-II was purified over 7000-fold with a final yield of 2.6%. By utilizing mass spectra analysis we can prove its first in-gel detection showing a migration pattern behavior that confirms its in silico molecular weight of 95.8 kDa. We could determine a turnover number of 2.18 min(-1) or one transferred xylose molecule per one XylT-II molecule each 27.5s. The k(cat)/K(M) ratio was 0.357 min(-1)microM(-1) for XylT-II using the bikunin-homologous acceptor Bio-QEEEGSGGGQKK-F. The comparison to XylT-I derived from the same organism revealed a 2.4-fold higher catalytic efficiency (0.870 min(-1)microM(-1)) for XylT-I.

Expression, purification and preliminary X-ray diffraction studies of the transcriptional factor PyrR from Bacillus halodurans.

The PyrR transcriptional regulator is widely distributed in bacteria. This RNA-binding protein is involved in the control of genes involved in pyrimidine biosynthesis, in which uridyl and guanyl nucleotides function as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bacillus halodurans PyrR are reported. One of the forms belongs to the monoclinic space group P2(1) with unit-cell parameters a = 59.7, b = 87.4, c = 72.1 A, beta = 104.4 degrees , while the other form belongs to the orthorhombic space group P22(1)2(1) with unit-cell parameters a = 72.7, b = 95.9, c = 177.1 A. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of four and six monomers per asymmetric unit; a crystallographic tetramer is formed in both forms.

Heterologous expression and biochemical characterization of soluble human xylosyltransferase II.

Human xylosyltransferase II (EC 2.4.2.26, XT-II) represents an isoform of xylosyltransferase I (XT-I). Recently, we and others provided first evidence that XT-II is capable of initiating the biosynthesis of glycosaminoglycan chains in proteoglycans. Here, a soluble form of human XT-II was expressed in the yeast Pichia pastoris and the substrate specificity for various acceptors was investigated, pointing to a modified bikunin peptide to be the optimal XT-II acceptor (K(M)=1.9 microM). Furthermore, biochemical characterization of XT-II showed that this enzyme was strongly inhibited by nucleotides and glycosaminoglycans. Its temperature optimum, stability, and ion dependency were further examined, demonstrating necessity for Mg(2+) or Mn(2+) ions for its enzymatic activity. Our data show for the first time that XT-I and XT-II are xylosyltransferases with similar but not identical properties, pointing to their potential role in modulating the cellular proteoglycan pool.

[Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-I].

AIM: To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity. METHODS: The predominant epitope of XT-I gene was predicted by the DNAssist software. The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli ER2566 and the fusion protein GST-XT was induced and isolated. The purified fusion protein was used to immunize New Zealand rabbits. The antibody titer was determined by ELISA. Purified polyclonal antibody was obtained through affinity chromatography column and the specificity of the purified antibody was characterized by Western blot. RESULTS: The amino acid 175-205 of XT-I (QKHQPELAKKPPSRQK-ELLKRKLEQQEKGKG) was selected as an antigen epitope. The synthesized DNA fragment of XT-I was successfully inserted into pGEX-4T-2 vector and the protein GST-XT was expressed. The purified fusion protein GST-XT was used as the immunogen to immunize rabbits and the polyclonal antibody against XT-I protein was obtained. The result of ELISA showed that the antibody titer was 1:640 000. Western blot analysis showed that the antibody had a good specificity. CONCLUSION: The rabbit polyclonal antibody against human XT-I protein has been successfully prepared, which lays the foundation for further study on the biosynthesis of PG by neoplastic myoepithelial cells in salivary tumors.

Crystallization of human nicotinamide phosphoribosyltransferase.

In the NAD biosynthetic pathway, nicotinamide phosphoribosyltransferase (NMPRTase; EC 2.4.2.12) plays an important role in catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide and 5'-phosphoribosyl-1'-pyrophosphate. Because the diffraction pattern of the initially obtained crystals was not suitable for structure analysis, the crystal quality was improved by successive use of the microseeding technique. The resultant crystals diffracted to 2.0 A resolution. These crystals belonged to space group P21, with unit-cell parameters a = 60.56, b = 106.40, c = 82.78 A. Here, the crystallization of human NMPRTase is reported in the free form; the crystals should be useful for inhibitor-soaking experiments on the enzyme.