Evolution of vitamin B2 biosynthesis: eubacterial RibG and fungal Rib2 deaminases.

Eubacterial RibG and yeast Rib2 possess a deaminase domain for pyrimidine deamination in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for ribose and ribitol, respectively. Here, the crystal structure of Bacillus subtilis RibG in complex with a deaminase product is reported at 2.56 Å resolution. Two loops move towards the product on substrate binding, resulting in interactions with the ribosyl and phosphate groups and significant conformational changes. The product carbonyl moiety is bent out of the pyrimidine ring to coordinate to the catalytic zinc ion. Such distortions in the bound substrate and product may play an essential role in enzyme catalysis. The yeast Rib2 structure was modelled and a mutational analysis was carried out in order to understand the mechanism of substrate recognition in these two enzymes. Detailed structural comparisons revealed that the two consecutive carbonyl backbones that occur prior to the PCXXC signature constitute a binding hole for the target amino group of the substrate. This amino-binding hole is essential in B. subtilis RibG and is also conserved in the RNA/DNA-editing deaminases.

Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis.

The non-Hodgkin lymphoma (NHL) subtype anaplastic large-cell lymphoma (ALCL) is frequently associated with a t(2;5)(p23;q35) that results in the fusion of the ubiquitously expressed nucleophosmin (NPM) gene at 5q35 to the anaplastic lymphoma kinase (ALK) gene at 2p23, which is not normally expressed in hematopoietic tissues. Approximately 20% of ALCLs that express ALK do not contain the t(2;5), suggesting that other genetic abnormalities can result in aberrant ALK expression. Here we report the molecular characterization of an alternative genetic means of ALK activation, the inv(2)(p23q35). This recurrent abnormality produces a fusion of the amino-terminus of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional homodimeric enzyme that catalyzes the penultimate and final steps of de novo purine nucleotide biosynthesis, with the intracellular portion of the ALK receptor tyrosine kinase. RT-PCR analysis of 5 ALCL tumors that contained the inv(2) revealed identical ATIC-ALK fusion cDNA junctions in all of the cases. Transient expression studies show that the ATIC-ALK fusion transcript directs the synthesis of an approximately 87-kd chimeric protein that is localized to the cytoplasm, in contrast to NPM-ALK, which typically exhibits a cytoplasmic and nuclear subcellular distribution. ATIC-ALK was constitutively tyrosine phosphorylated and could convert the IL-3-dependent murine hematopoietic cell line BaF3 to cytokine-independent growth. Our studies demonstrate an alternative mechanism for ALK involvement in the genesis of NHL and suggest that ATIC-ALK activation results from ATIC-mediated homodimerization. In addition, expected decreases in ATIC enzymatic function in ATIC-ALK-containing lymphomas may render these tumors more sensitive to antifolate drugs such as methotrexate. (Blood. 2000;95:2144-2149)

Structure and functional relationships in human pur H.

1. The human pur H (ATIC) gene encoding a bifunctional protein, hPurH, which carries the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway, AICARFT & IMPCH, has been cloned and sequenced. The gene product, hPurH has been overexpressed in E. coli, purified to homogeneity and crystallized. 2. The human pur H gene lies on chromosome 2, between band q34 and q35. There is at least one intron of 278 bp near the 5' end. 3. Truncation mutant studies demonstrate two non-overlapping functional domains in the protein arranged as indicated in Figure 5. The existence of a linker or interaction region between the catalytic domains remains to be established. 4. Cleland-type kinetic inhibition experiments indicate that the AICARFT reaction is of the ordered, sequential type with the reduced folate cofactor binding first. 5. The reaction has a broad pH optimum in the alkaline range, with a maximum at about pH 8.2. 6. Preliminary transient phase kinetic studies show the presence of a "burst" indicating that a late step in the reaction sequence is rate limiting. 7. A PurH crystal structure is that of a dimer, with a putative single binding site for the reduced folate cofactor formed using elements from each of the monomer subunits. Probable binding sites for AICAR and FAICAR can be identified on each monomer. 8. Equilibrium sedimentation studies show hPurH apoprotein to be a monomer:dimer equilibrium mixture with a kD of 0.55 uM. 9. The crystal structure has permitted identification of a number of candidate amino acid residues likely to be involved in catalysis and/or substrate binding. Among these, we have thus far completed studies on two, Lysine 265 and Histidine 266. These appear to be critically involved in the AICARFT reaction, although whether their role(s) are in catalysis or binding remains to be determined.

Inactivation of blasticidin S by Bacillus cereus. VI. Structure and comparison of the bsr gene from a blasticidin S-resistant Bacillus cereus.

Two types of recombinant plasmids containing 600 bp Nde I fragments that coded the bsr gene in opposite directions were obtained. Nucleotide sequencing shows that the bsr encodes a 140 amino acid protein with a putative molecular weight of 15560, the same as that of purified blasticidin S (BS)-deaminase (BSR), on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15500). Upstream of the open reading frame, a Shine-Dalgarno (SD) sequence, frequent inverted repeats, and the sigmaA and sigmaB promoter sequences are observed. The transcriptional start point was determined to be the A located 7 bases downstream from the putative sigmaA promoter (91TTGATC and 113TAAAAT) by the primer extension method and site directed mutagenesis at the -10 or -35 promoter region. A comparison of the amino acid sequence of BSR with that of BS-deaminase from Aspergillus terreus (BSD) showed 27.2% homology. Low degrees of homology were also observed with cytidine deaminase and deoxy cytidine monophosphate (CMP) deaminase. Four conserved amino acid motifs were observed, VGAx6G, C(orH)AEx6A, SPCGxCR, and Gx8ELIP (x(n) indicates a nonspecific residue and its position). It is possible that the three Cys residues and the Glu in the conserved motifs comprise the active center. Site-directed mutagenesis of the Cys residues supports this possibility.

De novo purine nucleotide biosynthesis: cloning, sequencing and expression of a chicken PurH cDNA encoding 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase-IMP cyclohydrolase.

The purH cDNA, encoding 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) transformylase-inosine monophosphate cyclohydrolase (ATIC), was cloned by functional complementation of an Escherichia coli purH mutant using a chicken liver cDNA expression library. This represents the first report of the cloning of any eukaryotic ATIC-encoding cDNA (PurH). The avian ATIC mRNA is 2.3 kb long and encodes a protein with an Mr of 64,422. The deduced amino acid sequence is 36% identical to the bacterial purH-encoded enzymes from Bacillus subtilis and E. coli. The avian cDNA was expressed as a glutathione S-transferase (GST) fusion protein that was purified in a single step by affinity chromatography. A novel vector was employed which permits rapid and highly efficient cleavage of the GST fusion protein yielding 10 mg of purified PurH product per liter of bacterial culture. Km values were determined with the purified fusion protein utilizing AICAR and (6-R)N10-formyl-tetrahydrofolate as substrates. These values compare favorably with the isolated avian enzyme, supporting the idea that kinetic, as well as other physical properties of the recombinant fusion protein are similar to the native avian enzyme. Large quantities of purified enzyme and the ability to generate site-directed mutations should make mechanistic studies possible. The recombinant enzyme also affords a simple and reliable approach to identifying new antifolates.

Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus.

The de novo purine biosynthetic enzymes 5-amino-4-imidazolecarboxamide-ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide-ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr 57,335 and 45,945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57,335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR transformylase and IMP cyclohydrolase activities. The 5' end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming 'hairpin' loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.

Evidence for sequential expression of multiple AMP deaminase isoforms during skeletal muscle development.

AMP deaminase (myoadenylate deaminase; EC 3.5.4.6) is an integral part of the myofibril in skeletal muscle, and this enzyme plays an important role in energy metabolism in this tissue. We report here the identification of three AMP deaminase isoforms during skeletal muscle development in the rat. An embryonic isoform is expressed in the developing hindlimb of the rat between 7 and 14 days of gestation. This isoform is not unique to skeletal muscle or the embryo as it is also expressed in many nonmuscle tissues of the perinatal and adult rat. A perinatal isoform of AMP deaminase that is restricted to skeletal muscle is produced 4-6 days before birth and persists for 2-3 weeks of postnatal life. An adult, skeletal muscle-specific isoform of AMP deaminase appears at birth and reaches maximal levels after 3 weeks of postnatal development. We conclude from these studies there is a developmentally controlled program that leads to the sequential expression of AMP deaminase isoforms during the transition from embryonic to adult skeletal muscle.

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.

5-methyl-dCTP deaminase induced by bacteriophage XP-12.

Bacteriophage XP-12, whose DNA contains 34 mol% 5-methylcytosine, induces the synthesis of a unique enzyme, 5-methyl-dCTP deaminase. The substrate for this enzyme, 5-methyl-dCTP, is produced by reactions catalyzed in part by other phage-induced enzymes, and the product of the reaction is dTTP. The deaminase therefore provides a novel pathway for biosynthesis of thymine residues for phage XP-12 DNA. Evidence is presented that this pathway is used for dTTP synthesis in XP-12-infected Xanthomonas oryzae.

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus.

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.

Bromodeoxyuridine induction of deoxycytidine deaminase activity in a hamster cell line.

The Syrian hamster cell line, RPMI 3460, was found to express barely detectable levels of the enzyme deoxycytidine deaminase. In contrast, the cell lines B4 and HAB, which are derived from 3460 cells and have approx. 60 and 100% bromodeoxyuridine substitution in DNA, respectively, show an approx. 50-fold higher enzyme activity. Deoxycytidine deaminase activity can be "induced" in 3460 cells by growth in 10(-5) M bromodeoxyuridine, as well as by the other halogenated pyrimidines, iododeoxyuridine and chlorodeoxy-uridine. The time required for maximal enzyme activity to accrue (approx. 8 days) suggests that new genetic expression is required for enhanced deoxycytidine deaminase activity and inhibition of induction in the presence of Ara. C shows that bromodeoxyuridine must be incorporated into DNA. In addition, the extent of enhanced deoxycytidine deaminase activity is directly related to the level of bromodeoxyuridine substitution in DNA. Another hamster cell line, BHK21/C13, which shows no detectable deoxycytidine deaminase activity, cannot be induced by bromodeoxyuridine. These results are discussed with respect to a mechanism by which bromodeoxyuridine may alter gene expression due to an altered binding of both positive and negative regulatory proteins to DNA.

Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene.

Deletion of the Escherichia coli K-12 chromosome associated with P2 mediated education extend through the structural gene for uridine kinase, udk, and the dcd gene encoding 2'-deoxycytidine 5'-triphosphate deaminase. The lack of uridine kinase makes a positive selection possible for these strains. Due to the dcd mutation, P2 eductants show large alterations in their deoxyribonucleoside triphosphate pools.

Bacteriophage PBS2-induced deoxycytidine triphosphate deaminase in Bacillus subtilis.

The dCTP deaminase induced by Bacillus subtilis bacteriophage PBS2, whose DNA contains uracil instead of thymine, requires metal ion and thiol activators and has a molecular weight of 125,000. The enzyme displays sigmoidal substrate saturation kinetics and inhibition by dUTP, consistent with the deaminase's proposed role of providing balanced levels of dUTP and dCTP for PBS2 uracil-DNA synthesis.