Purification and characterization of a bifunctional L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) from Rhodopseudomonas sphaeroides Y

Abstract

A bifunctional enzyme, L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1..., respectively), was discovered in cells of Rhodopseudomonas sphaeroides Y, which accounts for the ability of this organism to grow on L-(+)-tartrate and D-(+)-malate. The enzyme was purified 110-fold to homogeneity with a yield of 51%. During the course of purification, including ion-exchange chromatography and preparative gel electrophoresis, both enzyme activities appeared to be in association. The ratio of their activities remained almost constant [1:10, L-(+)-tartrate dehydrogenase/D-(+)malate dehydrogenase (decarboxylating)] throughout all steps of purification. Analysis by polyacrylamide gel electrophoresis revealed the presence of a single protein band, the position of which was coincident with both L-(+)-tartrate dehydrogenase and D-(+)-malate dehydrogenase (decarboxylating) activities. The apparent molecular weight of the enzyme was determined to be 158,000 by gel filtration and 162,000 by ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 38,500, indicating that the enzyme consisted of four subunits of identical size. The isoelectric point of the enzyme was between pH 5.0 and 5.2. The enzyme catalyzed the NAD-linked oxidation of L-(+)-tartrate as well as the oxidative decarboxylation of D-(+)-malate. For both reactions, the optimal pH was in a range from 8.4 to 9.0. The activation energy of the reaction (AlP) was 71.8 kJ/mol for L-(+)-tartrate and 54.6 kJ/mol for D-(+)-malate. NAD was required as a cosubstrate, and optimal activity depended on the presence of both

A Lysine-Tyrosine Pair Carries Out Acid−Base Chemistry in the Metal Ion-Dependent Pyridine Dinucleotide-Linked β-Hydroxyacid Oxidative Decarboxylases

“…(The protonated base, K199, could also stabilize the enolate without a formal proton transfer, but the pK a of the enolate is higher than that of K199, favoring proton transfer to form the enol.) In agreement, kinetic 13 C isotope effects measured for divalent metal ion-catalyzed decarboxylation of oxaloacetate are very similar to the intrinsic 13 C kinetic isotope effects for decarboxylation of the oxaloacetate intermediate in the malic enzyme reaction, suggesting the enzyme simply provides the site for binding metal ion and reactant and plays only a small catalytic role in this step of the reaction (57).…”

“…Deuterium isotope effects of unity on V and V/K HIc , obtained with homocitrate-2-D, indicate hydride transfer does not contribute to rate limitation (40); a small 13 (V/K HIc ) of 1.0057 was measured in the same study. Given the value of ∼1.05 estimated for the intrinsic 13 C isotope effect in the malic enzyme reaction, and assuming a similar value in the homoisocitrate dehydrogenase reaction, the chemical step must be at least 10-fold faster than a physical step along the reaction pathway. Solvent deuterium isotope effects suggest this step is a conformational change of the E-NAD-MgHIc complex.…”

“…tartrate dehydrogenase (12,13) require both a monovalent ion (usually K + ) and a divalent metal ion for optimal activity. Finally, 6-phosphogluconate dehydrogenase is divalent and monovalent metal ion-independent (14,15).…”

Aerobic L-tartrate Utilization by Bacillus Isolates

“…The cells were then subjected to sonication (VibraCell TM , SONICS, USA) for a total period of 3-5 minutes at a pulse rate of 10 s in an ice bath, followed by centrifugation at 8000xg at 4 °C for 30 minutes to remove cell debris. The resulting supernatant was used as the cell-free extract to determine TDH activity by following initial rate of NADH formation at 340 nm 7 . The final volume of 1 ml assay system contained 100 mM Tris-Cl buffer (pH-8.5), 1 mM b-mercaptoethanol, 0.4 mM MnCl 2 , 50 mM (NH 4 ) 2 SO 4 , 1.8 mM NAD and appropriate amount of crude cell lysate as enzyme source.…”

Screening for microorganisms producing D-malate from maleate

“…In the second degradation pathway, maleate is directly hydrated to D-malate (17,27). D-Malate is degraded by several microorganisms via an inducible, NAD+-dependent D-malic enzyme (14,16,(18)(19)(20)33). This enzyme catalyzes the oxidative decarboxylation of D-malate to pyruvate and CO2.…”