Tài liệu Báo cáo khoa học: Modulation of the enzymatic efficiency of ferredoxin-NADP(H) reductase by

Modulation of the enzymatic efficiency of

ferredoxin-NADP(H) reductase by the amino acid

volume around the catalytic site

Matı´as A. Musumeci, Adria´n K. Arakaki, Daniela V. Rial, Daniela L. Catalano-Dupuy and

Eduardo A. Ceccarelli

Molecular Biology Division, Instituto de Biologı´a Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioquı´micas y Farmace´uticas,

Universidad Nacional de Rosario, Argentina

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs,

EC 1.18.1.2) are a widely distributed class of flavoen￾zymes that have non-covalently bound FAD cofactor

as a redox center. FNRs participate in a wide variety

of redox-based metabolic reactions, transferring elec￾trons between obligatory one- and two-electron carri￾ers and therefore functioning as a general electron

splitter. In non-phototrophic bacteria and eukaryotes,

the reaction is driven towards ferredoxin (Fd) reduc￾tion, providing reducing power for multiple metabolic

pathways, including steroid hydroxylation in mamma￾lian mitochondria, nitrite reduction and glutamate

synthesis in heterotrophic tissues of vascular plants,

radical propagation and scavenging in prokaryotes,

and hydrogen and nitrogen fixation in anaerobes (for

a review, see [1,2]). In plants, FNR participates in

photosynthetic electron transport, reducing Fd at the

level of photosystem I, and transferring electrons to

NADP+. This process ends with the formation of the

NADPH necessary for CO2 fixation and other biosyn￾thetic pathways [2].

The three-dimensional structures of several FNRs

have been determined. They display similar structural

features, which have been defined as the prototype for

a large family of flavoenzymes [3–10]. Plant-type FNRs

can be classified into a plastidic class, characterized by

Keywords

catalytic efficiency; enzyme evolution;

ferredoxin; ferredoxin-NADP(H) reductase;

oxidoreductases

Correspondence

E. A. Ceccarelli, Molecular Biology Division,

Instituto de Biologı´a Molecular y Celular de

Rosario (IBR), CONICET, Facultad de

Ciencias Bioquı´micas y Farmace´uticas,

Universidad Nacional de Rosario, Suipacha

531, S2002LRK Rosario, Argentina

Fax: +54 341 4390465

Tel: +54 341 4351235

E-mail: [email protected]

(Received 1 November 2007, revised 8

January 2008, accepted 16 January 2008)

doi:10.1111/j.1742-4658.2008.06298.x

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous

flavoenzymes that deliver NADPH or low-potential one-electron donors

(ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in

plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient

reductases. In contrast, FNRs from organisms possessing a heterotrophic

metabolism or anoxygenic photosynthesis display turnover numbers 20- to

100-fold lower than those of their plastidic and cyanobacterial counterparts.

Several structural features of these enzymes have yet to be explained. The

residue Y308 in pea FNR is stacked nearly parallel to the re-face of the fla￾vin and is highly conserved amongst members of the family. By computing

the relative free energy for the lumiflavin–phenol pair at different angles

with the relative position found for Y308 in pea FNR, it can be concluded

that this amino acid is constrained against the isoalloxazine. This effect is

probably caused by amino acids C266 and L268, which face the other side

of this tyrosine. Simple and double FNR mutants of these amino acids were

obtained and characterized. It was observed that a decrease or increase in

the amino acid volume resulted in a decrease in the catalytic efficiency of

the enzyme without altering the protein structure. Our results provide exper￾imental evidence that the volume of these amino acids participates in the

fine-tuning of the catalytic efficiency of the enzyme.

Abbreviations

Fd, ferredoxin; Fld, flavodoxin; FNR, ferredoxin (flavodoxin)-NADP(H) reductase; IPTG, isopropyl thio-b-D-galactoside.

1350 FEBS Journal 275 (2008) 1350–1366 ª 2008 The Authors Journal compilation ª 2008 FEBS