Báo cáo khoa họcRe-engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in

Re-engineering the discrimination between the oxidized

coenzymes NAD+ and NADP+ in clostridial glutamate

dehydrogenase and a thorough reappraisal of the

coenzyme specificity of the wild-type enzyme

Marina Capone*, David Scanlon, Joanna Griffin and Paul C. Engel

School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland

Introduction

The nicotinamide-nucleotide-dependent dehydrogenases

tend, in general, to be either NAD+-specific (and then

catabolic) or NADP(H)-specific (and accordingly ana￾bolic, except for those few enzymes such as glucose

6-phosphate dehydrogenase which provide NADPH

for biosynthesis) [1]. Crystallographic studies of arche￾typal NAD+-specific enzymes, such as alcohol and

lactate dehydrogenases [2,3], and archetypal NADPH￾specific dehydrogenases such as glutathione reductase

[4] have offered some degree of understanding of the

ways in which these enzymes achieve their coenzyme

specificity. This has been augmented by various

detailed studies of amino acid sequences [5,6], and has

been both tested and applied in some notably success￾ful examples of re-engineering of coenzyme specificity

[7–19]. As noted by Khouri et al. [17], however, the

Keywords

burst kinetics; coenzyme purity; coenzyme

specificity; glutamate dehydrogenase;

site-directed mutagenesis

Correspondence

P. C. Engel, School of Biomolecular and

Biomedical Science, Conway Institute,

University College Dublin, Belfield,

Dublin 4, Ireland

Fax: +353 1 716 6456

Tel: +353 1 716 6764

E-mail: [email protected]

Present address

*Kuros Biosurgery AG, Zu¨rich, Switzerland

Program in Neurosciences & Mental

Health, Hospital for Sick Children, Toronto,

Canada

(Received 5 March 2011, revised 21 April

2011, accepted 9 May 2011)

doi:10.1111/j.1742-4658.2011.08172.x

Clostridial glutamate dehydrogenase mutants, designed to accommodate

the 2¢-phosphate of disfavoured NADPH, showed the expected large speci￾ficity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofac￾tors initially revealed little improvement with NADP+, although rates with

NAD+ were markedly diminished. This article reveals that the enzyme’s

discrimination in favour of NAD+ and against NADP+ had been greatly

underestimated and has indeed been abated by a factor of > 16 000 by the

mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed

a burst increase of A340 with NADP+ but not NAD+, with amplitude

depending on the concentration of the coenzyme, rather than enzyme.

Amplitude also varied with the commercial source of the NADP+. FPLC,

HPLC and mass spectrometry identified NAD+ contamination ranging

from 0.04 to 0.37% in different commercial samples. It is now clear that

apparent rates of NADP+ utilization mainly reflected the reduction of con￾taminating NAD+, creating an entirely false view of the initial coenzyme

specificity and also of the effects of mutagenesis. Purification of

the NADP+ eliminated the burst. With freshly purified NADP+, the

NAD+ : NADP+ activity ratio under standard conditions, previously esti￾mated as 300 : 1, is 11 000. The catalytic efficiency ratio is even higher at

80 000. Retested with pure cofactor, mutants showed marked specificity

shifts in the expected direction, for example, 16 200 fold change in catalytic

efficiency ratio for the mutant F238S ⁄ P262S, confirming that the key struc￾tural determinants of specificity have been successfully identified. Of wider

significance, these results underline that, without purification, even the best

commercial coenzyme preparations are inadequate for such studies.

2460 FEBS Journal 278 (2011) 2460–2468 ª 2011 The Authors Journal compilation ª 2011 FEBS