Structure-Function Analysis of 2-Sulfamoylacetic Acid Synthase in Altemicidin Biosynthesis

Academic Background

Sulfonamide antibiotics, such as altemicidin and its analogs, have garnered significant attention due to their notable antitumor and antibacterial activities. These compounds feature a rare sulfonamide side chain, which holds great importance in drug design and medical applications. However, the structure-activity relationship of the sulfonamide side chain in altemicidin and its analogs has not been fully elucidated. To further understand the function of this critical structure and its biosynthetic mechanism, researchers conducted an in-depth study on a key enzyme in the altemicidin biosynthetic pathway—2-sulfamoylacetic acid synthase (SbzJ).

SbzJ is an aldehyde dehydrogenase responsible for converting 2-sulfamoylacetic aldehyde into 2-sulfamoylacetic acid, a crucial step in generating the sulfonamide side chain. Although the function of SbzJ has been preliminarily confirmed, its substrate specificity and the structural basis for sulfonamide group recognition remain unclear. Therefore, this study aims to reveal the catalytic mechanism and substrate recognition properties of SbzJ through biochemical characterization and structure-function analysis, providing a theoretical foundation for future enzyme engineering and the development of novel bioactive compounds.

Source of the Paper

This paper was co-authored by Takahiro Mori, Kosuke Sakurada, Takayoshi Awakawa, Haibin He, Richiro Ushimaru, and Ikuro Abe, affiliated with the Graduate School of Pharmaceutical Sciences at the University of Tokyo, the Collaborative Research Institute for Innovative Microbiology at the University of Tokyo, the Japan Science and Technology Agency (JST), and the RIKEN Center for Sustainable Resource Science. The paper was published in 2024 in The Journal of Antibiotics, with the DOI 10.1038/s41429-024-00798-0.

Research Process and Results

1. Biochemical Characterization and Substrate Specificity Analysis of SbzJ

The study began with a systematic analysis of SbzJ’s substrate specificity through in vitro experiments. The results showed that SbzJ not only catalyzes the oxidation of its natural substrate, 2-sulfamoylacetic aldehyde (4), but also accepts various other aldehyde substrates, including butyraldehyde (6), 2-ethylbutyraldehyde (7), benzaldehyde (8), 2-phenylpropionaldehyde (9), cinnamaldehyde (10), and 3-methylthiobutyraldehyde (11). These substrates were oxidized by SbzJ to generate the corresponding carboxylic acids (12-17).

Through steady-state kinetic analysis, the researchers further quantified the catalytic efficiency of SbzJ for different substrates. The results revealed that SbzJ’s catalytic efficiency for 2-sulfamoylacetic aldehyde (kcat/Km = 6.2 × 10^4 s^-1 M^-1) is comparable to that of several known aldehyde dehydrogenases. Additionally, SbzJ exhibited high catalytic efficiency for butyraldehyde and 3-methylthiobutyraldehyde, indicating a preference for aliphatic and smaller aldehyde substrates.

2. Crystal Structure Determination of SbzJ

To elucidate the catalytic mechanism of SbzJ, the researchers determined the crystal structure of SbzJ in complex with NAD+ at a resolution of 2.5 Å. Structural analysis revealed that SbzJ adopts the typical aldehyde dehydrogenase fold, consisting of an NAD+-binding domain and a C-terminal catalytic domain. NAD+ binds to the active site through a hydrogen bond network, with His431 and Glu240 identified as key residues responsible for activating the catalytic Cys273 and a water molecule.

Using a molecular docking model, the researchers further simulated the binding mode of SbzJ with its substrate, 2-sulfamoylacetic aldehyde. The model showed that the sulfonamide group interacts with Tyr148, Ser272, and Gln425 through hydrogen bonds, indicating that SbzJ recognizes the sulfonamide group via hydrogen bonding. This finding provides a structural basis for SbzJ’s substrate specificity.

3. Mutagenesis Experiments to Validate Key Residues

To validate the function of the active site residues in SbzJ, the researchers conducted systematic mutagenesis experiments. The results showed that mutation of the catalytic residue Cys273 (C273A) completely abolished SbzJ’s oxidation activity. Additionally, mutations of Tyr148, Glu240, and His431 significantly reduced the enzyme’s activity, indicating that these residues play crucial roles in substrate recognition and catalysis. In particular, Tyr148, through hydrogen bonding with the sulfonamide group, is essential for substrate recognition.

4. Proposed Catalytic Mechanism

Based on structural analysis and mutagenesis results, the researchers proposed a catalytic mechanism for SbzJ: First, Cys273, activated by His431, attacks the aldehyde substrate to form a covalently bound thiohemiacetal intermediate. Subsequently, the hydride from the intermediate is transferred to the nicotinamide ring of NAD+, generating a thioester enzyme intermediate and NADH. Finally, a water molecule, assisted by His431 and Glu240, attacks the thioester bond, releasing the carboxylic acid product and regenerating free Cys273.

Research Conclusions and Significance

This study comprehensively revealed the catalytic mechanism and substrate specificity of SbzJ through crystal structure determination, in vitro enzymatic assays, and mutagenesis experiments. The findings demonstrate that SbzJ recognizes the sulfonamide group through a hydrogen bond network and utilizes key residues Tyr148, Glu240, and His431 to achieve efficient catalysis. These insights not only deepen the understanding of SbzJ’s function but also provide important clues for future enzyme engineering and the development of novel bioactive compounds.

Research Highlights

1. Broad Substrate Specificity: SbzJ can catalyze the oxidation of various aldehyde substrates, demonstrating broad substrate specificity.
2. Structural Mechanism Elucidation: Crystal structure determination revealed the structural basis for SbzJ’s recognition of the sulfonamide group.
3. Identification of Key Residues: Mutagenesis experiments confirmed the critical roles of Tyr148, Glu240, and His431 in catalysis.
4. Proposed Catalytic Mechanism: A catalytic mechanism for SbzJ was proposed, providing theoretical support for future enzyme engineering.

Additional Valuable Information

The crystal structure data from this study have been deposited in the Protein Data Bank (PDB) under the accession code 9JU5. Additionally, the research was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the New Energy and Industrial Technology Development Organization (NEDO), and the Japan Science and Technology Agency (JST).

Through this study, the researchers not only elucidated the catalytic mechanism of SbzJ but also provided new research directions for the biosynthesis of sulfonamide antibiotics and enzyme engineering. Future studies can leverage the structural characteristics of SbzJ to design enzyme variants with higher catalytic efficiency or novel substrate specificity, thereby developing more potent bioactive compounds.