PDBsum entry 3i4c

Oxidoreductase

PDB id: 3i4c

Contents

Protein chains

340 a.a. *

312 a.a. *

Metals

_ZN ×12

Waters ×429

* Residue conservation analysis

PDB id:

3i4c

Name:

Oxidoreductase

Title:

Crystal structure of sulfolobus solfataricus adh(ssadh) double mutant (w95l,n249y)

Structure:

NAD-dependent alcohol dehydrogenase. Chain: a, b, c, d, e, h. Engineered: yes. Mutation: yes

Source:

Sulfolobus solfataricus. Organism_taxid: 2287. Gene: adh, sso2536. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.228 R-free: 0.250

Authors:

L.Esposito,A.Pennacchio,A.Zagari,M.Rossi,C.A.Raia

Key ref:

A.Pennacchio et al. (2009). Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase. Extremophiles, 13, 751-761. PubMed id: 19588068

Date:

01-Jul-09 Release date: 21-Jul-09

Protein chains

P39462  (ADH_SULSO) -  NAD-dependent alcohol dehydrogenase from Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)

Seq: 347 a.a.

Struc: 340 a.a.*

Protein chains

P39462  (ADH_SULSO) -  NAD-dependent alcohol dehydrogenase from Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)

Seq: 347 a.a.

Struc: 312 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B, C, D, E, H: E.C.1.1.1.1 - alcohol dehydrogenase.
• Reaction:
  1. a primary alcohol + NAD⁺ = an aldehyde + NADH + H⁺
  2. a secondary alcohol + NAD⁺ = a ketone + NADH + H⁺
• Cofactor: Zn(2+) or Fe cation

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

reference

Extremophiles 13:751-761 (2009)

PubMed id: 19588068

Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

A.Pennacchio, L.Esposito, A.Zagari, M.Rossi, C.A.Raia.

ABSTRACT

A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.