Research group led by Professor Yoshimitsu Kakuta, Associate Professor Etsuko Nishimoto, and Assistant Professor Takamasa Teramoto elucidates the structure and function of KfiA, an enzyme involved in heparosan biosynthesis

Revealing how the structure of a glycan-elongating enzyme supports glycosyl transfer

---

Highlights

• Heparosan is a bacterial capsular polysaccharide with the same backbone as the biosynthetic precursor of animal heparan sulfate. The structural basis of the function of KfiA, an enzyme involved in its synthesis, has remained unclear.
• The researchers determined the crystal structure of KfiA and, together with molecular modeling, provided insights into the molecular mechanism underlying heparosan chain elongation.
• These findings deepen our understanding of heparosan chain elongation and are expected to provide a foundation for future research on enzymatic glycan synthesis.

---

Overview

A research group including Professor Yoshimitsu Kakuta, Associate Professor Etsuko Nishimoto, and Assistant Professor Takamasa Teramoto of the Faculty of Agriculture, Kyushu University; Kento Maeda, Aya Sakamoto, and Kota Toyoda of the Graduate School of Bioresource and Bioenvironmental Sciences; and Kyoka Matsuura of the Faculty of Agriculture (Laboratory of Biophysical Chemistry), together with collaborators within and outside Kyushu University, investigated the three-dimensional structure and enzymatic function of KfiA, an enzyme involved in the synthesis of heparosan produced by Escherichia coli K5.

Heparosan is a linear polysaccharide consisting of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) residues and shares its carbohydrate backbone with animal heparan sulfate. In E. coli K5, KfiA adds GlcNAc and another enzyme, KfiC, adds GlcA to the end of the growing chain, thereby extending heparosan. However, the structural basis by which KfiA recognizes its sugar donor and promotes efficient glycosyl transfer has not been fully understood.

In this study, the researchers determined X-ray crystal structures of a C-terminally truncated KfiA in its ligand-free state and in a state bound to UDP and a metal ion. The UDP-bound structure was resolved at 1.68 Å, revealing the characteristic GT-A fold and the binding site for UDP and the metal ion. Structure-guided mutational analyses further indicated that Asp81, Arg165, and Asp168 contribute to efficient utilization of the sugar donor, whereas Trp204 and the C-terminal region are particularly important for efficient transfer of sugars to the growing glycan chain. In addition, the researchers constructed an AlphaFold3 model of the KfiA-KfiC complex and proposed a possible molecular arrangement through which the two enzymes cooperate in glycan elongation.

These findings provide an important molecular framework for understanding how successive sugar residues are added during heparosan biosynthesis. Future studies may clarify how KfiA and KfiC cooperate and facilitate research on structure-guided enzymatic glycan synthesis.

The research was published in the Elsevier journal Carbohydrate Research on October 7, 2026.

---

Figure 1

---

Glossary

Heparosan: A linear polysaccharide composed of alternating GlcNAc and GlcA residues. It is a capsular polysaccharide of E. coli K5 and has the same carbohydrate backbone as the biosynthetic precursor of animal heparan sulfate.

KfiA / KfiC: Glycosyltransferases involved in heparosan biosynthesis in E. coli K5. KfiA adds GlcNAc, whereas KfiC adds GlcA to the growing chain.

UDP: Uridine diphosphate, one of the products released following glycosyl transfer.

X-ray crystallography: A technique for determining molecular three-dimensional structures at atomic resolution from X-ray diffraction data collected from protein crystals.

AlphaFold3: A computational method for predicting three-dimensional structures of proteins and complexes containing other molecules.

---

Acknowledgments

This work was supported by JSPS KAKENHI (21570115, 25440029, 24K09353, and 25H01276). X-ray diffraction experiments were supported at SPring-8 beamline BL26B1.

---

Publication Information

Journal: Carbohydrate Research
Title: Structural and functional analysis of KfiA, a UDP-GlcNAc transferase involved in Escherichia coli K5 heparosan biosynthesis
Authors: Kento Maeda, Aya Sakamoto, Kyoka Matsuura, Kota Toyoda, Hiroka Hori, Momo Onishi, Yuuichi Baba, Yoshirou Kawaguchi, Yohei Ishibashi, Nozomu Okino, Etsuko Nishimoto, Nobuo Sugiura, Takamasa Teramoto, Yoshimitsu Kakuta*
* Corresponding author
DOI: 10.1016/j.carres.2026.110123

---

Contact

Assistant Professor Takamasa Teramoto
Associate Professor Etsuko Nishimoto
Professor Yoshimitsu Kakuta
Laboratory of Biophysical Chemistry, Division of Applied Life Chemistry