A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides

Abstract

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases fromThermus thermophilusHB8

“…Recently, Esipov et al (2016) have described APRT from T. thermophilus HB27, which is identical to putative TtAPRT1 from T. thermophilus HB8. Since TtAPRT2 is also encoded in T. thermophilus HB27 genome, it leads to think that two different APRTS are present in T. thermophilus, the first one, TtAPRT1, displays an activity value of 8.8 lM min À1 mg À1 (referred to AMP synthesis) (Esipov et al 2016), while TtAPRT2 display an activity of 8000 lMmin À1 mg À1 (deduced from Table 1). In this sense, it seems that TtAPRT2 should play a more important role in purine scavenging in T. thermophilus HB8 than TtAPRT1.…”

“…Due to this, the use of PRTs as therapeutic targets has been extensively reported (el Kouni 2003). In contrast, only a few examples about their use as catalysts in NMP synthesis has been described (Scism et al 2007(Scism et al , 2010Valino et al 2015;Iglesias et al 2015;Esipov et al 2016;Hansen et al 2014).…”

“…According to their substrate specificity, purine PRTs are classified into two general classes: 6-aminopurine PRTs (APRT), strictly specific for 6-aminopurines, such as adenine and 6-aminopurine derivatives (Craig and Eakin 2000;Iglesias et al 2015;Esipov et al 2016), and 6-oxopurine PRTs (HPRT, GPRT, XPRT, HGPRT, XGPRT or HGXPRT) that can recognize different kinds of 6-oxopurines, such as hypoxanthine, guanine, xanthine and other 6-oxopurine analogs (el Kouni 2003;Craig and Eakin 2000;Scism et al 2007;Scism & Bachmann 2010;Iglesias et al 2015;Valino et al 2015).…”

Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives

“…Tt HB8 APRT was shown to be active and stable in a pH range of 5-6, and in a temperature range from 60 to 90°C (optimal activity pH 6 and 70-80°C). More recently, the substrate specificity of an APRT from T. thermophilus HB27 (Tt HB27 APRT) has been tested (Esipov et al 2016). Tt HB27 APRT displays activity towards different 6aminopurine derivatives, such as 2,6-diaminopurine, 2fluoroadenine, 2-chloradenine, 2-methoxyadenine, or N1methyladenine (Fig.…”

“…6). The production of 2-fluoroadenosine-5′-monophosphate (≈ 37%) and 2-chloroadenosine-5′monophosphate (≈ 30%) from D-ribose and the corresponding nucleobase was carried out by this thermophilic cascade system (Esipov et al 2016) In 6-oxopurine PRTs, the oligomerization state seems to play a key role not only in catalysis but also in both stabilization of the active conformation and overall protein stability. Thermostable 6-oxopurine PRTs are described in the literature as dimers and tetramers (Del Arco et al 2017c, Del Arco andFernández-Lucas 2017;Hansen et al 2014;Sinha and Smith 2001), as illustrated by dimeric HGXPRT from S. solfataricus and tetrameric HGXPRT from T. thermophilus.…”