3U1V

X-ray Structure of De Novo design cysteine esterase FR29, Northeast Structural Genomics Consortium Target OR52

Summary for 3U1V

• Entry DOI: 10.2210/pdb3u1v/pdb
• Related: 3FHJ
• Descriptor: De Novo design cysteine esterase FR29 (2 entities in total)
• Functional Keywords: structural genomics, psi-biology, protein structure initiative, northeast structural genomics consortium, nesg, fr29, de novo design, unknown function
• Biological source: synthetic construct
• Total number of polymer chains: 4
• Total formula weight: 155387.67

Authors

Kuzin, A.,Su, M.,Vorobiev, S.M.,Seetharaman, J.,Patel, D.,Xiao, R.,Ciccosanti, C.,Richter, F.,Everett, J.K.,Acton, T.B.,Baker, D.,Montelione, G.T.,Hunt, J.F.,Tong, L.,Northeast Structural Genomics Consortium (NESG) (deposition date: 2011-09-30, release date: 2011-12-07, Last modification date: 2024-10-30)

Primary citation

Richter, F.,Blomberg, R.,Khare, S.D.,Kiss, G.,Kuzin, A.P.,Smith, A.J.,Gallaher, J.,Pianowski, Z.,Helgeson, R.C.,Grjasnow, A.,Xiao, R.,Seetharaman, J.,Su, M.,Vorobiev, S.,Lew, S.,Forouhar, F.,Kornhaber, G.J.,Hunt, J.F.,Montelione, G.T.,Tong, L.,Houk, K.N.,Hilvert, D.,Baker, D.
Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.
J.Am.Chem.Soc., 134:16197-16206, 2012

PubMed Abstract: Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

PubMed: 22871159
DOI: 10.1021/ja3037367

Experimental method

X-RAY DIFFRACTION (2.797 Å)