Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase.

An FMN-dependent NADH-azoreductase of Escherichia coli was purified and analyzed for identification of the gene responsible for azo reduction by microorganisms. The N-terminal sequence of the azoreductase conformed to that of the acpD gene product, acyl carrier protein phosphodiesterase. Overexpression of the acpD gene provided the E. coli with a large amount of the 23-kDa protein and more than 800 times higher azoreductase activity. The purified gene product exhibited activity corresponding to that of the native azoreductase. The reaction followed a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red (4'-dimethylaminoazobenzene-2-carboxylic acid) into 2-aminobenzoic acid and N,N'-dimethyl-p-phenylenediamine. On the other hand, the gene product could not convert holo-acyl carrier protein into the apo form under either in vitro or in vivo conditions. These data indicate that the acpD gene product is not acyl carrier protein phosphodiesterase but an azoreductase.

Purification and properties of recombinant Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase.

Recombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized. Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat values. The values of both forward and reverse reactions of P. falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme. Km values of the parasite and human SAH enzymes are 1.2 and 7.8 microM, respectively. On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P. falciparum, are 101 nM for the parasite enzyme and 47 nM for human enzyme. P. falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria. This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.

2-Methyladenosine-Substituted 2',5'-oligoadenylates: conformations, 2-5A binding and catalytic activities with human ribonuclease L.

2-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me2A), p5'(me2A)2'p5'A2'p5'A, and p5'(me2A) 2'p5'(me2A)2'pS'(me2A), were prepared via a modification of a lead ion-catalyzed ligation reaction. These 5'-monophosphates were subsequently converted into the corresponding 5'-triphosphates. Both binding and activation of human recombinant RNase L by various 2-methyladenosine-substituted 2-5A analogues were examined. Among the 2-5A analogues, p5'A2'p5'A2'p5'(me2A) showed the strongest binding affinity and was as effective as 2-5A itself as an activator of RNase L. The CD spectra of both p5'(me2A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(me2A) were superimposable on that of p5'A2'p5'A2'p5'A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2-5A.

Synthesis of 2-5As possessing base-modified adenosines and their activities to human recombinant RNase L.

The unique 2',5'-oligoadenylate (2-5A) acts as a potent inhibitor of translation in vertebrate cells through the activation of a constituent latent 2-5A-dependent endoribonuclease (RNase L). This 2-5A system plays a major role in the interferon natural defense mechanism against viral infection. We report the syntheses of base-modified adenosine-substituted 2-5A derivatives, their interaction with recombinant human RNase L and their biological stability.

Synthesis of carbocyclic nucleosides and their SAH hydrolase inhibitory activities.

The cellular enzyme S-adenosyl-L-homocysteine (SAH) hydrolase has emerged as a target enzyme for the molecular design of anti-viral agents. Recently, SAH hydrolase has been considered as an attractive target in parasite chemotherapy for malaria. We report synthesis of several carbocyclic purine nucleosides and their inhibitory activities against human and malaria recombinant SAH hydrolases.

9-[(2'S,3'S)-3'-formyl-2',3'-dihydroxypropyl]adenine: a facile affinity-labeling probe of human S-adenosyl-L-homocysteine hydrolase.

Treatment of human recombinant S-adenosyl-L-homocysteine (SAH) hydrolase with 9-[(2'S,3'S)-3'-formyl-2',3'-dihydroxypropyl]adenine (FDHPA) caused irreversible inactivation in a time- and concentration-dependent manner (Ki = 8.8 microM, k(inact) = 0.09 min(-1)). FDHPA behaved as a facile affinity-labeling probe of SAH hydrolase.

Synthesis of S-adenosyl-L-homocysteine hydrolase inhibitors and their biological activities.

Several nucleosides have been prepared as a possible inhibitor of human S-adenosyl-L-homocysteine (SAH) hydrolase for the development of anti-viral agents. Recently, SAH hydrolase has been considered as an attractive target for parasite chemotherapy for malaria. We report synthesis of several nucleosides including carbocyclic nucleosides and their inhibitory activities against recombinant malaria and human SAH hydrolases.

Facile method for the preparation of 7-methyl-8-oxoguanosines as an immunomodulator.

Reaction of 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-7-methylguaninium iodide (2a) with hydrogen peroxide in acetic acid gave the corresponding 7-methyl-8-oxoguanosine derivative (3a) in good yield. Deprotection of 3a easily gave 7-methyl-8-oxoguanosine (1), which is well-known as an immunomodulator. Substitution of acetyl group at the N2-position of guanine ring accelerated the oxidation reaction of the 7-methylguaninium iodide.

Synthesis of DHPA analogs and their inhibitory activities of human recombinant S-adenosyl-L-homocysteine hydrolase.

We report syntheses of DHPA analogs, e.g., 9-(3-formyl-2,3-dihydroxypropyl)adenine (FDHPA) and 9-(3-formyl-2,3-dihydroxypropyl)hypoxanthine (FDHPI). Among these DHPA analogs, FDHPA behaved as an irreversible inactivator of human recombinant SAH hydrolase.