Snapshots of PLP‐substrate and PLP‐product external aldimines as intermediates in two types of cysteine desulfurase enzymes

• Published in: The FEBS journal Vol. 287; no. 6; pp. 1138 - 1154
• Main Authors: Nakamura, Ryosuke, Hikita, Masahide, Ogawa, Shoko, Takahashi, Yasuhiro, Fujishiro, Takashi
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.03.2020
• Subjects: Bacillus subtilis - enzymology; Biocatalysis; Biomolecules; Biosynthesis; Carbon-Sulfur Lyases - chemistry; Carbon-Sulfur Lyases - metabolism; Catalysis; Crystallography; Crystallography, X-Ray; Cysteine; cysteine desulfurase; Data bases; enzyme catalysis; Enzymes; Helicobacter pylori - enzymology; Histidine; Imines; Imines - chemistry; Imines - metabolism; Intermediates; Iron; Models, Molecular; Pyridoxal Phosphate - chemistry; Pyridoxal Phosphate - metabolism; reaction intermediates; Substrates; Sulfur; X‐ray crystallography; X-ray crystallography; enzyme catalysis; cysteine desulfurase; reaction intermediates; biosynthesis
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.15081

Cysteine desulfurase enzymes catalyze sulfur mobilization from l‐cysteine to sulfur‐containing biomolecules such as iron‐sulfur (Fe‐S) clusters and thio‐tRNAs. The enzymes utilize the cofactor pyridoxal‐5'‐phosphate (PLP), which forms the external substrate‐ and product‐aldimines and ketimines during catalysis and are grouped into two types (I and II) based on their different catalytic loops. To clarify the structure‐based catalytic mechanisms for each group, we determined the structures of the external substrate‐ and product‐aldimines as catalytic intermediates of NifS (type I) and SufS (type II) that are involved in Fe‐S cluster biosynthesis using X‐ray crystallographic snapshot analysis. As a common intermediate structure, the thiol group of the PLP‐l‐cysteine external aldimine is stabilized by the conserved histidine adjacent to PLP through a polar interaction. This interaction makes the thiol group orientated for subsequent nucleophilic attack by a conserved cysteine residue on the catalytic loop in the state of PLP‐l‐cysteine ketimine, which is formed from the PLP‐l‐cysteine aldimine. Unlike the intermediates, structural changes of the loops were different between the type I and II enzymes. In the type I enzyme, conformational and topological change of the loop is necessary for nucleophilic attack by the cysteine. In contrast, the loop in type II cysteine desulfurase enzymes showed no large conformational change; rather, it might possibly orient the thiol group of the catalytic cysteine for nucleophilic attack toward PLP‐l‐cysteine. The present structures allow a revision of the catalytic mechanism and may provide a clue for consideration of enzyme function, structural diversity, and evolution of cysteine desulfurase enzymes. Database Structural data are available in PDB database under the accession numbers 5WT2, 5WT4, 5ZSP, 5ZST, 5ZS9, 5ZSK, 5ZSO, 6KFZ, 6KG0, and 6KG1. Catalytic reaction intermediates of two distinct types of PLP‐dependent cysteine desulfurase enzymes, NifS (type I) and SufS (type II), involved in iron‐sulfur cluster biosynthesis were structurally captured by X‐ray crystallographic snapshot analysis. The present data confirmed that NifS and SufS utilized the common PLP‐external aldimine intermediates. Also, it was demonstrated that they used their characteristic catalytic loops in different ways during NifS and SufS catalysis.