Ma Ming's team has made new progress in the structure and catalytic mechanism of furanose biosynthetase

On January 12, 2023, the Ma Ming research team of the State Key Laboratory of Natural Medicine and Biomimetic Drugs of our hospital published a title at ACS Catalysis "Structural Insight into a Metal-Dependent Mutase Revealing an Arginine Residue-Covalently Mediated Interconversion between Nucleotide-Based Pyranose and Furanose" research paper
。 This study elucidates the crystal structure of the first ion-dependent mutase in furanose ring biosynthesis and reveals an arginine covalently binding substrate-mediated coconversion mechanism of the pyranose ring and the furanose ring
.

Furanose (five-membered ring) usually has worse thermodynamic preference than the corresponding pyranose (six-membered ring), but in nature, furanose is an important structural unit of cell wall and pectin in bacteria, fungi, plants, such as gafuranose is the component unit of bacterial cell wall polysaccharides, and arabifuranose is the component unit
of plant pectin.
In addition to being the building blocks of primary metabolites, furanose is also a raw material for the synthesis of important natural products, such as galactofuran and fucofurose, which are the biosynthetic units
of antibiotics A201A and hygromycin A, respectively.

In view of the widespread existence and important role of furanose in nature, the biosynthetic mechanism of how furanose is formed in nature is of interest in both primary and secondary metabolism
.
The current study shows that these furanoses are formed by the ring shrinkage reaction of their corresponding pyranose, and the biosynthetic enzyme that catalyzes this cycle shrinkage reaction is called mutase
.
Mutases catalyze the interconversion of nucleoside linking pyranose and furanose , and two classes of mutases have been reported: flavin-dependent mutases (with flavin FAD or FMN as cofactors) and ion-dependent mutases (with divalent ions such as ^Mg2+).
and ^Mn2+ as cofactors).

More than 50 three-dimensional structures of flavin-dependent mutants have been published by PDBs, and their catalytic mechanisms have been elucidated (flavin cofactor covalently binding substrate-mediated cycloshrink reactions), while no ion-dependent mutase structures and catalytic mechanisms have been reported
.

Our research team has studied the structure and catalytic mechanism of an ion-dependent mutant (MtdL) in the biosynthesis of natural product A201A.

The crystal structure of the parent body and MtdL-GDP-Mg2⁺ complex of MtdL was analyzed, and it was found that the structure of the enzyme was quite different
from the published enzyme structure in PDB.
The most similar structure of MtdL in Dali's three-dimensional structure alignment analysis is the structure of the glycosyltransferase GlfT2 (glutinosyltransferase furanolactase in the cell wall of Mycobacteriaceae), but the similarity is still poor, and the two are similar only in the structure Fragment
of "Rossmann-like fold".
Needless to say, the three-dimensional structure of MtdL is completely different from that of flavin-dependent mutases, suggesting that they have different catalytic mechanisms
.

Based on the crystal structure of the MtdL-GDP-Mg2⁺ complex, the research team studied the catalytic mechanism through molecular dynamics simulation
.
Molecular dynamics simulations of substrates, intermediates, and products revealed important amino acid residues at the active site, and it was speculated that one of the arginine Arg159 could mediate the conversion
of pyranose to furanose by covalently binding the substrate.
Site-directed mutation experiments on Arg159 and other residues verify their important functions
.
In order to confirm the catalytic mode of Arg159 covalently binding substrate, MtdL was incubated with substrate, hydrolyzed by trypsin, and then analyzed
by mass spectrometry proteomics.
The results showed that a peptide containing Arg159 gave a series of fragment ions, all of which clearly pointed to the substrate covalently binding to Arg159, thereby supporting the catalytic mechanism
of MtdL.
MtdL was sequence-aligned with other ion-dependent mutases (such as UAMs responsible for arabinofuranose biosynthesis in plant pectin in primary metabolism), and it was found that Arg159 and other active residues were preserved.
It suggests that these ion-dependent mutases may have a catalytic mechanism
similar to that of MtdL.

In conclusion, this study elucidates for the first time the structure and catalytic mechanism of ion-dependent mutases in furanose biosynthesis, which provides a basis
for the engineering modification of such enzymes and the enzymatic preparation of furanoses.
Another highlight of the study is that enzymes that use arginine as a key residue to catalyze reactions are common, but they usually use arginine's ability to form hydrogen bonds and salt bridges for catalysis; Enzymes catalyzed by covalently binding substrates using arginine are rare
.
Therefore, the revelation of the catalytic mechanism of MtdL in this study also increases the understanding
of the role of arginine in biochemical reactions.

This work was supported
by the Key Research and Development Program of the Ministry of Science and Technology and the National Natural Science Foundation of China.
Chi Changbiao, a postdoctoral fellow at Peking University School of Pharmacy (currently working in the Department of Bioengineering and Technology, Huaqiao University), is the first author of this paper, and Ma Ming, a researcher at Peking University School of Pharmacy, and Li Qinglian, a researcher at the South China Sea Institute of Oceanology, Chinese Academy of Sciences, are co-corresponding authors
.
This study is the preliminary work of Ju Jianhua's team from the South China Sea Institute of Oceanology, Chinese Academy of Sciences (PNAS, 2017, 114, 4948-4953; Organic Letters, 2018, 20, 1015-1018.
Thank you for the great support of researcher Ju Jianhua!

Original link: https://doi.
org/10.
1021/acscatal.
2c04907

First author:

Chi Changbiao postdoctoral fellow

About the corresponding author:

Ma Ming is a researcher at the State Key Laboratory of Natural Medicines and Biomimetic Drugs, School of Pharmacy, Peking University, doctoral supervisor, and long-term associate professor
.
His research interests include biosynthesis of natural medicinal molecules, three-dimensional structure of enzymes and their catalytic mechanisms, and synthetic biology
of natural products.
For the past 5 years, he has been the corresponding author in Angew.
Chem.
Int.
Ed 、JACS、ACS Catal.
、Chem.
Sci.
、Acta Pharm.
Sin.
B.
、Commun.
Chem.
、Org.
Lett.
、ACS Chem.
Biol.
、J.
Biol.
Chem.
、J.
Org.
Chem.
、J.
Nat.
Prod.
and other international academic journals published 18 papers
.
He is a member of the Marine Drug Professional Committee of the Chinese Pharmaceutical Association, a member of the Youth Committee of the Chinese Association of Chinese Medicine, and a member of the Natural Medicine Professional Committee of
the Beijing Pharmaceutical Association.

Contributed by State Key Laboratory of Natural Medicines and Biomimetic Drugs