PDBsum entry 3fpc

Oxidoreductase

PDB id: 3fpc

Contents

Protein chains

352 a.a. *

Ligands

CAC ×4

OXY

EDO ×14

NO3 ×2

IMD

Metals

_NA ×2

_ZN ×4

Waters ×1988

* Residue conservation analysis

PDB id:

3fpc

Name:

Oxidoreductase

Title:

Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-294 of t. Brockii adh by e. Histolytica adh

Structure:

NADP-dependent alcohol dehydrogenase. Chain: a, b, c, d. Engineered: yes. Other_details: chimera of alcohol dehydrogenase by exchange of cofactor binding domain res 153-294 of thermoanaerobacter brockii by entamoeba histolytica

Source:

Thermoanaerobacter brockii, entamoeba histolytica. Thermoanaerobium brockii. Organism_taxid: 29323, 5759. Gene: adh1, adh1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.40Å R-factor: 0.118 R-free: 0.155

Authors:

F.Felix,E.Goihberg,L.Shimon,Y.Burstein

Key ref:

E.Goihberg et al. (2010). Biochemical and structural properties of chimeras constructed by exchange of cofactor-binding domains in alcohol dehydrogenases from thermophilic and mesophilic microorganisms. Biochemistry, 49, 1943-1953. PubMed id: 20102159

Date:

05-Jan-09 Release date: 19-Jan-10

Protein chains

P14941  (ADH_THEBR) -  NADP-dependent isopropanol dehydrogenase from Thermoanaerobacter brockii

Seq: 352 a.a.

Struc: 352 a.a.*

Protein chains

P35630  (ADH1_ENTHI) -  NADP-dependent isopropanol dehydrogenase from Entamoeba histolytica (strain ATCC 30459 / HM-1:IMSS / ABRM)

Seq: 366 a.a.

Struc: 352 a.a.*

* PDB and UniProt seqs differ at 122 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.1.1.80 - isopropanol dehydrogenase (NADP(+)).
• Reaction: propan-2-ol + NADP⁺ = acetone + NADPH + H⁺

propan-2-ol + NADP(+) (Bound ligand (Het Group name = EDO) matches with 60.00% similarity) = acetone + NADPH + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Biochemistry 49:1943-1953 (2010)

PubMed id: 20102159

Biochemical and structural properties of chimeras constructed by exchange of cofactor-binding domains in alcohol dehydrogenases from thermophilic and mesophilic microorganisms.

E.Goihberg, M.Peretz, S.Tel-Or, O.Dym, L.Shimon, F.Frolow, Y.Burstein.

ABSTRACT

The cofactor-binding domains (residues 153-295) of the alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) have been exchanged. Three chimeras have been constructed. In the first chimera, the cofactor-binding domain of thermophilic TbADH was replaced with the cofactor-binding domain of its mesophilic counterpart CbADH [chimera Chi21((TCT))]. This domain exchange significantly destabilized the parent thermophilic enzyme (DeltaT(1/2) = -18 degrees C). The reverse exchange in CbADH [chimera Chi22((CTC))], however, had little effect on the thermal stability of the parent mesophilic protein. Furthermore, substituting the cofactor-binding domain of TbADH with the homologous domain of EhADH1 [chimera Chi23((TET))] substantially reduced the thermal stability of the thermophilic ADH (DeltaT(1/2) = -51 degrees C) and impeded the oligomerization of the enzyme. All three chimeric proteins and one of their site-directed mutants were crystallized, and their three-dimensional (3D) structures were determined. Comparison of the 3D structures of the chimeras and the chimeric mutant with the structures of their parent ADHs showed no significant changes to their Calpha chains, suggesting that the difference in the thermal stability of the three parent ADHs and their chimeric mutants could be due to a limited number of substitutions located at strategic positions, mainly at the oligomerization interfaces. Indeed, stabilization of the chimeras was achieved, to a significant extent, either by introduction of a proline residue at a strategic position in the major horse liver ADH-type dimerization interface (DeltaT(1/2) = 35 degrees C) or by introduction of intersubunit electrostatic interactions (DeltaT(1/2) = 6 degrees C).