Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl–fluoride inhibitor of serine peptidases

Rodríguez-Banqueri, Arturo; Moliner-Culubret, Marina; Mendes, Soraia R.; Guevara, Tibisay; Eckhard, Ulrich; Gomis-Rüth, F.X.

doi:10.1039/d3dt00458a

Dalton Trans.

2023

DOI: 10.1039/d3dt00458a

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Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl–fluoride inhibitor of serine peptidases

Arturo Rodríguez-Banqueri

Marina Moliner-Culubret

Soraia R. Mendes

et al.

Abstract: Peptidases are regulated by latency and inhibitors, as well as compatibilization and cofactors. Ulilysin from Methanosarcina acetivorans, also called lysargiNase, is an archaeal metallopeptidase (MP) that is biosynthesized as a...

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Dissecting Cytophagalysin: Structural and Biochemical Studies of a Bacterial Pappalysin-Family Metallopeptidase

Estevan-Morió,

Ramírez-Larrota,

Bushi

et al. 2024

Biomolecules

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Cytophaga is a genus of Gram-negative bacteria occurring in soil and the gut microbiome. It is closely related to pathogenic Flavobacterium spp. that cause severe diseases in fish. Cytophaga strain L43-1 secretes cytophagalysin (CPL1), a 137 kDa peptidase with reported collagenolytic and gelatinolytic activity. We performed highly-confident structure prediction calculations for CPL1, which identified 11 segments and domains, including a signal peptide for secretion, a prosegment (PS) for latency, a metallopeptidase (MP)-like catalytic domain (CD), and eight immunoglobulin (Ig)-like domains (D3–D10). In addition, two short linkers were found at the D8–D9 and D9–D10 junctions, and the structure would be crosslinked by four disulfide bonds. The CPL1 CD was found closest to ulilysin from Methanosarcina acetivorans, which assigns CPL1 to the lower-pappalysin family within the metzincin clan of MPs. Based on the structure predictions, we aimed to produce constructs spanning the full-length enzyme, as well as PS+CD, PS+CD+D3, and PS+CD+D3+D4. However, we were successful only with the latter three constructs. We could activate recombinant CPL1 by PS removal employing trypsin, and found that both zymogen and mature CPL1 were active in gelatin zymography and against a fluorogenic gelatin variant. This activity was ablated in a mutant, in which the catalytic glutamate described for lower pappalyins and other metzincins was replaced by alanine, and by a broad-spectrum metal chelator. Overall, these results proved that our recombinant CPL1 is a functional active MP, thus supporting the conclusions derived from the structure predictions.

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Dissecting Cytophagalysin: Structural and Biochemical Studies of a Bacterial Pappalysin-Family Metallopeptidase

Estevan-Morió,

Ramírez-Larrota,

Bushi

et al. 2024

Biomolecules

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Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl–fluoride inhibitor of serine peptidases

Abstract: Peptidases are regulated by latency and inhibitors, as well as compatibilization and cofactors. Ulilysin from Methanosarcina acetivorans, also called lysargiNase, is an archaeal metallopeptidase (MP) that is biosynthesized as a...

Cited by 1 publication

References 93 publications

Dissecting Cytophagalysin: Structural and Biochemical Studies of a Bacterial Pappalysin-Family Metallopeptidase

Dissecting Cytophagalysin: Structural and Biochemical Studies of a Bacterial Pappalysin-Family Metallopeptidase

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