Robert Sarfati

CMP Kinase from Escherichia coli Is Structurally Related to Other Nucleoside Monophosphate Kinases

CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6, have unit cell parameters a = b = 82.3 Å and c = 60.7 Å, and diffract x-rays to a 1.9 Å resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin‐activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13–24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site‐directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin‐binding properties of mutated proteins showed no important differences when compared to the wild‐type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3′‐anthraniloyl‐2′‐deoxyadenosine 5′‐triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP‐utilizing enzymes, are essential for binding the Mg(2+)‐nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide‐binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.

Role of nucleoside diphosphate kinase in the activation of anti-HIV nucleoside analogs.

Nucleoside analogs are currently used in antiretrovirus therapies. The best known example isAZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphatederivatives of these compounds act as substrates of the viral reverse transcriptase. Since theydo not enter cells, nucleoside analogs are administered and phosphorylated by cellular kinases.The last step in this phosphorylation pathway is catalyzed by nucleoside diphosphate (NDP)kinase. The incorporation of the nucleoside triphosphates into nascent viral DNA chain resultsin termination of the elongation process. We have performed kinetics studies of thephosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2′,3′-dideoxy-3′-azidothymidine diphosphate (AZT-DP) and 2′,3′-dideoxy-2′,3′-didehydrothymidinediphosphate (d4T-DP). We show that the catalytic efficiency is strongly decreased and, therefore,that the reaction step catalyzed by NDP kinase constitutes a bottleneck in the processingpathway of anti-HIV compounds. In addition, the affinity of the analogs in the absence ofcatalysis was determined using a catalytically inactive NDP kinase mutant, showing a reductionof affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinaseprovides a structural explanation for these results. Indeed, all nucleoside analogs acting aschain terminators must lack a 3′-OH in the nucleotide deoxyribose. Unfortunately, this samesubstitution is detrimental for their capacity to be phosphorylated by NDP kinase. This definesthe framework for the design of new nucleoside analogs with increased efficiency inantiretroviral therapies.

The Motif D Loop of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Critical for Nucleoside 5′-Triphosphate Selectivity

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the “palm” domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment fromTaq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3′-deoxy 3′-azidothymidine 5′-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.

Synthesis of fluorescent derivatives of 3′-O-(6-aminohexanoyl)pyrimidine nucleosides 5′-triphosphates that act as DNA polymerase substrates reversibly tagged at C-3′

Treatment, of 5′-O-dimethoxytritylthymidine 1 and of 4-N-benzyloxycarbonyl-2′-deoxy-5′-O-dimethoxy-trityl-cytidine 12 with a mixture of 6-(benzyloxycarbonylamino)hexanoic acid, dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine yielded 3′-O-acylated derivatives 2 and 13. These were further converted into 3′-O-(6-aminohexanoyl)thymidine 7 and 3′-O-(6-aminohexanoyl)-2′-deoxycytidine 16 monophosphates. The latter compounds were labelled at the aliphatic amino group with fluorescent probes and transformed into their triphosphates 9 and 18.

Enzymatic synthesis of 3′:5′ cyclic AMP using Bordetella pertussis adenylate cyclase co-immobilized with calmodulin on agarose beads

Abstract We report a simple and efficient enzymatic synthesis of 3′:5′ cAMP from ATP using Bordetella pertussis adenylate cyclase coimmobilized on CNBr-activated agarose beads with its activator, calmodulin. The bacterial adenylate cyclase was purified in a single step by chromatography on calmodulin-agarose. Immobilized adenylate cyclase/calmodulin complex lost only 50% of its activity after one month of storage at room temperature. The enzymatic synthesis of cAMP offers obvious advantages over chemical procedures. The reaction proceeds in a single step almost to completion and the purification and crystallization of cAMP (87% overall yield) is straightforward .

Borano-nucleotides: new analogues to circumvent HIV-1 RT-mediated nucleoside drug-resistance.

α-Boranophosphates suppress RT-mediated resistance when the catalytic rate of incorporation (kpol) of the analogue 5′-triphosphate is responsable for drug resistance, such as in the case of K65R mutant and ddNTPs, and Q151M toward AZTTP and ddNTPs. This suppression is also observed with BH3-d4T and BH3-3TC toward their clinically relevant mutants Q151M and M184V. Moreover, the presence of the borano (BH3 −) group renders the incorporation of the analogue independent from amino-acid substitutions in RT. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analogue.

Asparagine‐135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket

This work studies the structure‐function relationships of Asn135, a residue situated in the GTP binding pocket of elongation factor Tu (EF‐Tu). For this purpose we constructed EF‐TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF‐TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn135 side‐chain does not recognize the exocyclic keto group of the guanine base. However, EF‐TuN 135D/D 138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa‐tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn135 is important for the correct folding of the nucleotide binding site and that EF‐Tu·kirromycin can mediate the binding of aa‐tRNA to the mRNA‐programmed ribosomes independently of the native conformation of this site.

3'-MODIFIED NUCLEOTIDES FOR DNA SEQUENCING WITHOUT GEL ELECTROPHORESIS

Abstract Dibenzyl-3′-O-[(6-azido-2,3,6-trideoxy-4,5-di-O-benzyl) hexanoyl] thymidine 5′-yl phosphate 8a was prepared. Catalytic hydrogenolysis removed only the benzyl esters and reduced the azido group. When the benzyl ethers were replaced by p-phenylbenzyl or allyl ethers, their deprotection also failed.

New fluorescent and photoactivable analogs acting on nucleotide binding enzymes

Abstract We describe a seven-step synthesis of 8-azido-3′-O-anthraniloyl-2′dADP and 2′dATP from 8-azido-2′deoxyadenosine. These compounds can be used as fluorescent and photoactivable probes for the nucleotide-binding site of kinases or cyclases.