Tài liệu Báo cáo khoa học: Proton transfer in the oxidative half-reaction of pentaerythritol

Proton transfer in the oxidative half-reaction of

pentaerythritol tetranitrate reductase

Structure of the reduced enzyme-progesterone complex and the

roles of residues Tyr186, His181 and His184

Huma Khan1

, Terez Barna1

, Neil C. Bruce2

, Andrew W. Munro1,*, David Leys1,† and

Nigel S. Scrutton1,†

1 Department of Biochemistry, University of Leicester, UK

2 CNAP, Department of Biology, University of York, UK

Pentaerythritol tetranitrate (PETN) reductase was ori￾ginally purified from a strain of Enterobacter cloacae

(strain PB2) on the basis of its ability to utilize nitrate

ester explosives such as PETN and glycerol trinitrate

(GTN) as sole nitrogen source. Sequence analysis [1]

and structural studies [2] have indicated that PETN

reductase is a flavoprotein member of the Old Yellow

Enzyme (OYE) family [3]. Other well-defined members

include bacterial morphinone reductase (MR) from

Pseudomonas putida M10 [4], estrogen binding protein

Keywords

crystallography; flavoprotein mechanism;

kinetics; Old Yellow Enzyme; PETN

reductase

Correspondence

N. S. Scrutton, Faculty of Life Sciences,

University of Manchester, Stopford Building,

Oxford Road, Manchester, M13 9PT, UK

Fax: +44 161 2755586

Tel: +44 161 2755632

E-mail: [email protected]

Present addresses

*Manchester Interdisciplinary Biocentre,

School of Chemical Engineering and Ana￾lytical Science, University of Manchester,

The Mill, PO Box 88, Manchester,

M60 1QD, UK

†Manchester Interdisciplinary Biocentre and

Faculty of Life Sciences, Faculty of Life

Sciences, University of Manchester,

Stopford Building, Oxford Road,

Manchester, M13 9PT, UK

(Received 13 June 2005, revised 15 July

2005, accepted 21 July 2005)

doi:10.1111/j.1742-4658.2005.04875.x

The roles of His181, His184 and Tyr186 in PETN reductase have been

examined by mutagenesis, spectroscopic and stopped-flow kinetics, and by

determination of crystallographic structures for the Y186F PETN reductase

and reduced wild-type enzyme—progesterone complex. Residues His181

and His184 are important in the binding of coenzyme, steroids, nitro￾aromatic ligands and the substrate 2-cyclohexen-1-one. The H181A and

H184A enzymes retain activity in reductive and oxidative half-reactions,

and thus do not play an essential role in catalysis. Ligand binding and

catalysis is not substantially impaired in Y186F PETN reductase, which

contrasts with data for the equivalent mutation (Y196F) in Old Yellow

Enzyme. The structure of Y186F PETN reductase is identical to wild-type

enzyme, with the obvious exception of the mutation. We show in PETN

reductase that Tyr186 is not a key proton donor in the reduction of a ⁄ b

unsaturated carbonyl compounds. The structure of two electron-reduced

PETN reductase bound to the inhibitor progesterone mimics the catalytic

enzyme-steroid substrate complex and is similar to the structure of the

oxidized enzyme-inhibitor complex. The reactive C1-C2 unsaturated bond

of the steroid is inappropriately orientated with the flavin N5 atom for

hydride transfer. With steroid substrates, the productive conformation is

achieved by orientating the steroid through flipping by 180
, consistent

with known geometries for hydride transfer in flavoenzymes. Our data

highlight mechanistic differences between Old Yellow Enzyme and PETN

reductase and indicate that catalysis requires a metastable enzyme-steroid

complex and not the most stable complex observed in crystallographic

studies.

Abbreviations

EBP, estrogen binding protein; GTN, glycerol trinitrate; MR, morphinone reductase; OYE1, Old Yellow Enzyme 1; PETN, pentaerythritol

tetranitrate; TNT, trinitrotoluene.

4660 FEBS Journal 272 (2005) 4660–4671 ª 2005 FEBS