Purification and Characterization of Malate Synthase from the Glucose-grown Wood-rotting BasidiomyceteFomitopsis palustris

Abstract

Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was puriˆed to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The puriˆed enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have K m of 45 and 2.2 mM for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (K i , 8.5 mM) and glycolate (K i , 17 mM), and uncompetitively by coenzyme A (K i , 100 mM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg 2+ (K m , 0.4 mM), which also served to signiˆcantly prevent enzyme inactivation during storage.

Oxalate Efflux Transporter from the Brown Rot Fungus Fomitopsis palustris

“…Moreover, we have suggested that F. palustris transports oxalate out of the cells to prevent possible inhibition of intracellular metabolic reactions. For example, oxalate might inhibit the activities of FPICL1 and malate synthase in peroxisomes of F. palustris (15,35), because oxalate competitively inhibits the activities of the two enzymes in vitro (30,31). In addition, oxalate produced from oxaloacetate by cytosolic OAAH in F. palustris (1,35) possibly shows product inhibition for OAAH activity because oxalate is a competitive inhibitor (K i ϭ 19 M) for OAAH from Botrytis cinerea (13).…”

β-Carboline Alkaloids from Galianthe ramosa Inhibit Malate Synthase from Paracoccidioides spp.

“…80 % by HPLC), the MIC value was 0.270 µM. We evaluated the effects of alkaloids 1 and 2 on PbMLS using pyruvic acid as a positive control [18][19]. Alkaloid 1 exhibited no inhibitory activity against PbMLS, but alkaloid 2 inhibited PbMLS with an IC 50 of 50 µM (l " Fig.…”

Purification and Characterization of NADP-Linked Isocitrate Dehydrogenase from the Copper-tolerant Wood-rotting BasidiomyceteFomitopsis palustris

“…10) Moreover, we have reported that the metabolic change occurs at the transient step from the vegetative growth to the fruit body formation of this basidiomycete; ICL, a key enzyme of the GLOX cycle, is more important in oxalate synthesis at the vegetative growth stage, and NADP-IDH is important for glutamate synthesis at the fruiting stage. 11) Furthermore, we have recently puriˆed and characterized the malate synthase (MS) 12) and ICL 13) involved in the GLOX cycle of the same fungus.…”