Tài liệu Báo cáo khoa học: Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from

Cosubstrate-induced dynamics of D-3-hydroxybutyrate

dehydrogenase from Pseudomonas putida

Karthik S. Paithankar1

, Claudia Feller2

, E. Bartholomeus Kuettner1

, Antje Keim1

, Marlis Grunow2

and Norbert Stra¨ter1

1 Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy,

University of Leipzig, Germany

2 Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, University of Leipzig, Germany

Short-chain dehydrogenases⁄reductases (SDRs) consti￾tute a large protein family that now includes more

than 1000 enzymes in humans, mammals, insects and

bacteria [1]. The dehydrogenases act on a wide variety

of substrates, including steroids, retinoids, prostaglan￾dins, sugars and alcohols. The name SDR is based on

their smaller subunit size of about 250 residues com￾pared with the medium-chain dehydrogenase ⁄reductase

family that has a subunit size of about 350 residues.

The SDR enzymes exhibit a sequence identity of

15–30% and share two signature motifs: a GxxxGxG

motif involved in coenzyme binding; and a YxxxK

Keywords

crystal structure; loop closure; protein

dynamics; SDR

Correspondence

M. Grunow, Institute of Biochemistry,

Faculty of Biosciences, Pharmacy, and

Psychology, University of Leipzig,

Bru¨derstraße 34, D-04103 Leipzig, Germany

Fax: +49 341 9736998

Tel: +49 341 9736907

E-mail: [email protected]

N. Stra¨ter, Center for Biotechnology and

Biomedicine, Institute of Bioanalytical

Chemistry, Faculty of Chemistry and

Mineralogy, University of Leipzig, Deutscher

Platz 5, D-04103 Leipzig, Germany

Fax: +49 341 9731319

Tel: +49 341 9731311

E-mail: [email protected]

(Received 5 July 2007, revised 8 August

2007, accepted 10 September 2007)

doi:10.1111/j.1742-4658.2007.06102.x

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to

the family of short-chain dehydrogenases⁄reductases. We have determined

X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudo￾monas putida, which was recombinantly expressed in Escherichia coli, in

three different crystal forms to resolutions between 1.9 and 2.1 A˚ . The so￾called substrate-binding loop (residues 187–210) was partially disordered in

several subunits, in both the presence and absence of NAD+. However,

in two subunits, this loop was completely defined in an open conformation

in the apoenzyme and in a closed conformation in the complex structure

with NAD+. Structural comparisons indicated that the loop moves as a

rigid body by about 46. However, the two small a-helices (aFG1 and

aFG2) of the loop also re-orientated slightly during the conformational

change. Probably, the interactions of Val185, Thr187 and Leu189 with the

cosubstrate induced the conformational change. A model of the binding

mode of the substrate D-3-hydroxybutyrate indicated that the loop in the

closed conformation, as a result of NAD+ binding, is positioned compe￾tent for catalysis. Gln193 is the only residue of the substrate-binding loop

that interacts directly with the substrate. A translation, libration and screw

(TLS) analysis of the rigid body movement of the loop in the crystal

showed significant librational displacements, describing the coordinated

movement of the substrate-binding loop in the crystal. NAD+ binding

increased the flexibility of the substrate-binding loop and shifted the equi￾librium between the open and closed forms towards the closed form. The

finding that all NAD+-bound subunits are present in the closed form and

all NAD+-free subunits in the open form indicates that the loop closure is

induced by cosubstrate binding alone. This mechanism may contribute to

the sequential binding of cosubstrate followed by substrate.

Abbreviations

PfHBDH, Pseudomonas fragi D-3-hydroxybutyrate dehydrogenase; PpHBDH, Pseudomonas putida D-3-hydroxybutyrate dehydrogenase;

SDR, short-chain dehydrogenase ⁄ reductase.

FEBS Journal 274 (2007) 5767–5779 ª 2007 The Authors Journal compilation ª 2007 FEBS 5767