Tomoki Hamamoto

Enzymatic synthesis of 2'-deoxyguanosine with nucleoside deoxyribosyltransferase-II.

Nucleoside deoxyribosyltransferase-II (NdRT-II) of Lactobacillus helveticus, which catalyzes the transfer of a glycosyl residue from a donor deoxyribonucleoside to an acceptor base, has a broad specificity for the acceptor bases. Six-substituted purines were found to be substrates as acceptor bases for NdRT-II. Using this property of the enzyme, we established a practical procedure for enzymatic synthesis of 2′-deoxyguanosine (dGuo), consisting of the transglycosylation from thymidine to 6-substituted purine (2-amino-6-chloropurine; ACP) instead of natural guanine and the conversion of 2-amino-6-chloropurine-2′-deoxyriboside (ACPdR) to dGuo with bacterial adenosine deaminase. Through the successive reactions, dGuo was synthesized in high yield.

An Efficient Method for Production of Uridine 5′-Diphospho-N-Acetylglucosamine

Uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been synthesized by a yeast-based method from 5′-UMP and glucosamine, in which yeast cells catalyze the conversion of 5′-UMP to 5′-UTP and provide enzymes involved in UDP-GlcNAc synthesis using 5′-UTP and glucosamine as substrates. However, this conventional method is not suitable for practical production of UDP-GlcNAc because of the low yield of the product. We found that the yqgR gene product of Bacillus subtilis, which has been identified as a glucokinase, can catalyze the phosphorylation of N-acetylglucosamine (GlcNAc) to give GlcNAc-6-phosphate, an intermediate of UDP-GlcNAc biosynthesis. The addition of the yqgR gene product to the yeast-based reaction system enabled us to synthesize UDP-GlcNAc using GlcNAc in place of glucosamine. The addition of two enzymes, GlcNAc-phosphate mutase and UDP-GlcNAc pyrophosphorylase, increased the yield of UDP-GlcNAc. Using this novel method, UDP-GlcNAc was produced at an amount of 78 mM from 100 mM 5′-UMP and 100 mM GlcNAc.

Enzymatic Synthesis of Cytidine 5′-Monophospho-N-acetylneuraminic Acid

We have established an efficient method for enzymatic production of cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-NeuAc) from inexpensive materials, N-acetylglucosamine (GlcNAc) and cytidine 5′-monophosphate (CMP). The Haemophilus influenzae nanE gene encoding GlcNAc 6-phosphate (GlcNAc 6-P) 2-epimerase and the Campylobacter jejuni neuB1 gene encoding N-acetylneuraminic acid (NeuAc) synthetase, both of whose products are involved in NeuAc biosynthesis, were cloned and co-expressed in Escherichia coli cells. We examined the synthesis of NeuAc from GlcNAc via GlcNAc 6-P, N-acetylmannosamine (ManNAc) 6-P, and ManNAc by the use of E. coli cells producing GlcNAc 6-P 2-epimerase and NeuAc synthetase, in expectation of biological functions of E. coli such as the supply of phosphoenolpyruvate (PEP), which is an essential substrate for NeuAc synthetase, GlcNAc phospholylation by the PEP-dependent phosphotransferase system, and dephospholylation of ManNAc 6-P. Eleven mM NeuAc was synthesized from 50 mM GlcNAc by recombinant E. coli cells with the addition of glucose as an energy source. Next we attempted to synthesize CMP-NeuAc from GlcNAc and CMP using yeast cells, recombinant E. coli cells, and H. influenzae CMP-NeuAc synthetase, and succeeded in efficient production of CMP-NeuAc due to a sufficient supply of PEP and efficient conversion of CMP to cytidine 5′-triphosphate by yeast cells.