Identification of Asp258 as the Metal Coordinate of Pigeon Liver Malic Enzyme by Site-Specific Mutagenesis

Abstract

Pigeon liver malic enzyme was inactivated by ferrous sulfate in the presence of ascorbate. Manganese and some other divalent metal ions provided complete protection of the enzyme against the Fe(2+)-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at Asp258-Ile259, which was presumably the Mn(2+)-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., & Chang, G. G. (1994) Biochemistry 33, 7793-7936]. For identification of Asp258 as the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp258 with glutamate (D258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respectively. These mutant proteins were recombinantly expressed in a bacterial expression system (pET-15b) with a stretch of histidine residues attached at the N-terminus and were successfully purified to apparent homogeneity by a single Ni-chelated affinity column. Among the four mutants, only D258E possessed 0.8% residual activity after purification; all other purified mutants had < 0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susceptible to inactivation by the Fe(2+)-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser extent as compared to the wild-type enzyme. None of the mutants were cleaved by the Fe(2+)-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp258.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme

“…In pigeon liver malic enzyme, a Fe2+-ascorbate system inactivates the enzyme by cleavage at the peptide bond between Asp"' and Ile259 (Wei et al, 1997). Moreover, site-directed mutagenesis has confirmed that Aspz5' is one of the ligands of Mn" (Wei et al, 1995). Our results suggest that this consensus aspartate, and thus consensus site, plays the same role of metal-binding site not only in all malic enzymes but also in all malolactic enymes.…”

“…The functions of these sites have been intensively studied in malic enzymes by different enzymic methods and by site-directed mutagenesis (Chang et al, 1993;Gavva et al, 1991 ;Wei et al, 1995Wei et al, , 1997. The consensus box 111 was exactly identical between malic and all malolactic enzymes.…”

Selective Oxidative Modification and Affinity Cleavage of Pigeon Liver Malic Enzyme by the Cu2+-Ascorbate System

“…Without a three-dimensional crystal structure available, affinity cleavage at the putative metal-binding site by the metal-catalyzed oxidation system (MCO) 1 may be the optimal approach of reaching the above goal. Using this technique with the Fe 2ϩ -ascorbate system, Asp 258 is successfully identified in our previous study as one of the metal-binding sites (Wei et al, 1994), as confirmed by site-directed mutagenesis (Wei et al, 1995). In that study, some divalent metal ions were found to be capable of providing protection of the enzyme against Fe 2ϩ -induced inactivation.…”

Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein

“…12,35,36 The other metal ligands include a water molecule and the 2-hydroxyl group of L-malate. 34 L-malate was proposed to bind with the enzyme through a metal bridge, which serves as an electrophile in the activation of a substrate.…”