INVESTIGADORES
SANTOS Javier
artículos
Título:
Direct Cysteine Desulfurase Activity Determination by NMR and the Study of the Functional Role of Key Structural Elements of Human NFS1
Autor/es:
SEWELL, KARL E.; GOLA, GABRIEL F.; PIGNATARO, MARÍA FLORENCIA; HERRERA, MARÍA GEORGINA; NOGUERA, MARTÍN E.; OLMOS, JUSTO; RAMÍREZ, JAVIER A.; CAPECE, LUCIANA; ARAN, MARTÍN; SANTOS, JAVIER
Revista:
ACS CHEMICAL BIOLOGY
Editorial:
AMER CHEMICAL SOC
Referencias:
Año: 2023 vol. 18 p. 1534 - 1547
ISSN:
1554-8929
Resumen:
The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron-sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: ?GGG? and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron-sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron-sulfur cluster assembly site but also modulates the internal motions of ISCU2.