Esipov R S, Abramchik Yu A, Fateev I V, Konstantinova I D, Kostromina M A, Muravyova T I, Artemova K G, Miroshnikov A I
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya Str., 16/10, Moscow, GSP-7, 117997, Russia.
Acta Naturae. 2016 Oct-Dec;8(4):82-90.
We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of -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 . 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from HB27 into the expression vectors, generated high-yield producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of -pentoses into 5-phosphates that were further converted into 5-phospho-α--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 -ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.
我们提出了一种合成具有生物学重要性的核苷酸的新方法，该方法包括将戊糖多酶级联转化为嘌呤核苷酸。该方法利用了嗜热微生物的核酸交换酶：核糖激酶、磷酸核糖焦磷酸合成酶和腺嘌呤磷酸核糖转移酶。我们从2.9中克隆了核糖激酶基因，以及从HB27中克隆了磷酸核糖焦磷酸合成酶（PRPP合成酶）的两个不同基因和腺嘌呤磷酸核糖转移酶（APR转移酶）基因到表达载体中，构建了高产生产菌株，开发了酶的纯化方法，并研究了酶的底物特异性。这些酶用于将戊糖转化为5-磷酸酯，然后通过核糖激酶和PRPP合成酶进一步将其转化为磷酸-α-D-戊呋喃糖1-焦磷酸酯。通过在APR转移酶催化的反应中，焦磷酸酯与腺嘌呤及其衍生物缩合得到目标核苷酸。在ATP、核糖激酶、PRPP合成酶和APR转移酶存在的情况下，使用嗜热酶系统，从D-核糖和适当的杂环碱一锅法合成了2-氯和2-氟单磷酸腺苷。
