Basic residues play key roles in catalysis and NADP(+)-specificity in maize (Zea mays L.) photosynthetic NADP(+)-dependent malic enzyme.

C(4)-specific (photosynthetic) NADP(+)-dependent malic enzyme (NADP(+)-ME) has evolved from C(3)-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP(+)-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP(+)-ME K255I (Lys-255-->Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP(+)-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play 'back-up' roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP(+) versus NAD(+)) of maize NADP(+)-ME by interacting with the 2'-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP(+) or NAD(+), as well as by the reciprocal inhibition constants of the competitive inhibitors 2'-AMP and 5'-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436-->Ile) with wild-type NADP(+)-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP(+)-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.

[1]  P. Paneth,et al.  Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves , 1991, Photosynthesis Research.

[2]  Saigo Mariana,et al.  Maize recombinant non-C4 NADP-malic enzyme: A novel dimeric malic enzyme with high specific activity , 2004, Plant Molecular Biology.

[3]  L. Tong,et al.  Structure and function of malic enzymes, a new class of oxidative decarboxylases. , 2003, Biochemistry.

[4]  D. Coleman,et al.  Crystallographic Studies on Ascaris suum NAD-Malic Enzyme Bound to Reduced Cofactor and Identification of an Effector Site* , 2003, Journal of Biological Chemistry.

[5]  L. Tong,et al.  Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism. , 2003, Structure.

[6]  M. F. Drincovich,et al.  Maize C4 NADP-Malic Enzyme , 2003, The Journal of Biological Chemistry.

[7]  Liang Tong,et al.  Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate. , 2002, Structure.

[8]  E. Goldsmith,et al.  Crystal structure of the malic enzyme from Ascaris suum complexed with nicotinamide adenine dinucleotide at 2.3 A resolution. , 2002, Biochemistry.

[9]  L. Tong,et al.  Structural studies of the pigeon cytosolic NADP+‐dependent malic enzyme , 2002, Protein science : a publication of the Protein Society.

[10]  L. Tong,et al.  Determination of the mechanism of human malic enzyme with natural and alternate dinucleotides by isotope effects. , 2001, Archives of biochemistry and biophysics.

[11]  M. F. Drincovich,et al.  NADP‐malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways , 2001, FEBS letters.

[12]  P. Cook,et al.  Lysine 199 is the general acid in the NAD-malic enzyme reaction. , 2000, Biochemistry.

[13]  T. Chin,et al.  Lysine residues 162 and 340 are involved in the catalysis and coenzyme binding of NADP(+)-dependent malic enzyme from pigeon. , 2000, Biochemical and biophysical research communications.

[14]  Liang Tong,et al.  Structure of a closed form of human malic enzyme and implications for catalytic mechanism , 2000, Nature Structural Biology.

[15]  C. Hwang,et al.  Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis. , 1999, Biochemistry.

[16]  L. Tong,et al.  Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases. , 1999, Structure.

[17]  A. R. Ashton.  NADP-malic enzyme from the C4 plant Flaveria bidentis: nucleotide substrate specificity. , 1997, Archives of biochemistry and biophysics.

[18]  P. Cook,et al.  Stepwise versus concerted oxidative decarboxylation catalyzed by malic enzyme: a reinvestigation. , 1994, Biochemistry.

[19]  I. Mikaélian,et al.  A general and fast method to generate multiple site directed mutations. , 1992, Nucleic acids research.

[20]  G. Edwards,et al.  NAD‐malic enzyme from plants , 1985, Phytochemistry.

[21]  K. Sandhoff,et al.  A mutation in the gene of a glycolipid‐binding protein (GM2 activator) that causes GM2‐gangliosidosis variant AB , 1991, FEBS letters.

[22]  A. Bhagwat,et al.  Chemical modification of the functional arginine residue(s) of malic enzyme from Zea mays , 1991 .

[23]  R. Wedding,et al.  Purification of NAD malic enzyme from potato and investigation of some physical and kinetic properties. , 1981, Archives of biochemistry and biophysics.

[24]  J. Sedmak,et al.  A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. , 1977, Analytical biochemistry.

[25]  P. Fromageot,et al.  Crystallographic studies on luteoskyrine , 1969, FEBS letters.

[26]  L. Reed,et al.  MECHANISM OF ENZYMATIC OXIDATIVE DECARBOXYLATION OF PYRUVATE , 1953 .