PDBsum entry 3u1v

Structural genomics, unknown function

PDB id: 3u1v

Contents

Protein chains

327 a.a.

269 a.a.

Waters ×32

PDB id:

3u1v

Name:

Structural genomics, unknown function

Title:

X-ray structure of de novo design cysteine esterase fr29, northeast structural genomics consortium target or52

Structure:

De novo design cysteine esterase fr29. Chain: a, b, c, d. Engineered: yes

Source:

Synthetic construct. Organism_taxid: 32630

Resolution:

2.80Å R-factor: 0.218 R-free: 0.290

Authors:

A.Kuzin,M.Su,S.M.Vorobiev,J.Seetharaman,D.Patel,R.Xiao,C.Ciccosanti, F.Richter,J.K.Everett,T.B.Acton,D.Baker,G.T.Montelione,J.F.Hunt, L.Tong,Northeast Structural Genomics Consortium (Nesg)

Key ref:

F.Richter et al. (2012). Computational design of catalytic dyads and oxyanion holes for ester hydrolysis. J Am Chem Soc, 134, 16197-16206. PubMed id: 22871159

Date:

30-Sep-11 Release date: 07-Dec-11

Protein chains

No UniProt id for this chain

Struc: 327 a.a.

Protein chain

No UniProt id for this chain

Struc: 269 a.a.

J Am Chem Soc 134:16197-16206 (2012)

PubMed id: 22871159

Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.

F.Richter, R.Blomberg, S.D.Khare, G.Kiss, A.P.Kuzin, A.J.Smith, J.Gallaher, Z.Pianowski, R.C.Helgeson, A.Grjasnow, R.Xiao, J.Seetharaman, M.Su, S.Vorobiev, S.Lew, F.Forouhar, G.J.Kornhaber, J.F.Hunt, G.T.Montelione, L.Tong, K.N.Houk, D.Hilvert, D.Baker.

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.