Abstract：
:Imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima is a model enzyme for studying allostery. The ImGPS complex consists of the cyclase subunit HisF and the glutaminase subunit HisH whose activity is stimulated by substrate binding to HisF in a V-type manner. To investigate the significance of a putative closing hinge motion at the cyclase:glutaminase interface for HisH activity, we replaced residue W123 in HisH with the light-switchable unnatural amino acid phenylalanine-4'-azobenzene (AzoF). Crystal structure analysis employing angle, buried surface area, and distance measurements showed that incorporation of AzoF at this position causes a closing of the interface by ∼18 ± 3%. This slightly different interface configuration results in a much higher catalytic efficiency in unstimulated HisH due to an elevated turnover number. Moreover, the catalytic efficiency of HisH when stimulated by binding of a substrate to HisF was also significantly increased by AzoF incorporation. This was caused by a K-type stimulation that led to a decrease in the apparent dissociation constant for its substrate, glutamine. In addition, AzoF improved the apparent binding of a substrate analogue at the HisF active site. Remarkably, light-induced isomerization of AzoF considerably enhanced these effects. In conclusion, our findings confirm that signal transduction from HisF to HisH in ImGPS involves the closing of the cyclase:glutaminase subunit interface and that incorporation of AzoF at a hinge position reinforces this catalytically relevant conformational change.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Kneuttinger AC,Rajendran C,Simeth NA,Bruckmann A,König B,Sterner Rdoi
10.1021/acs.biochem.0c00332subject
Has Abstractpub_date
2020-07-28 00:00:00pages
2729-2742issue
29eissn
0006-2960issn
1520-4995journal_volume
59pub_type
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