Purification and Characterization of Maleate Hydratase from Pseudomonas pseudoalcaligenes

Abstract

Maleate hydratase (malease) from Pseudomonas pseudoalcaligenes has been purified. The purified enzyme (98% pure) catalyzes the stereospecific addition ofwater to maleate and citraconate (2-methylmaleate), forming D-(+)-malate and D-(+)-citramalate, respectively. 2,3-Dimethylmaleate was also a substrate for malease. The stability of the enzyme was dependent on the protein concentration and the addition of dicarboxylic acids. The purified enzyme (89 kDa) consisted of two subunits (57 and 24 kDa). No cofactor was required for full activity of this colorless enzyme. Maximum enzyme activity was measured at pH 8 and 45°C. The K,m for maleate was 0.35 mM, and that for citraconate was 0.20 mM. Thiol reagents, such as p-chloromercuribenzoate and iodoacetamide, and sodium dodecyl sulfate completely inhibited malease activity. Malease activity was competitively inhibited by D-malate (Ki = 0.63 mM) and D-citramalate (Ki = 0.083 mM) and by the substrate analog 2,2-dimethylsuccinate (Ki = 0.025 mM). The apparent equilibrium constants for the maleate,

Purification and characterization of a Baeyer‒Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways

“…Steady-state kinetic parameters were determined from incubations (1 ml ; 30.0 mC) containing 40 µg of MMKMO, 50 mM glycine\NaOH buffer (pH 9.5), 0.2 mM NADPH, and substrate at concentrations ranging between 0.05 and 1 mM. Kinetic parameters were calculated from Lineweaver-Burk plots, as described previously [15]. The substrate specificity of MMKMO was determined by replacing (4R)-dihydrocarvone in the standard activity assay with 1 mM of the other substrates.…”

The selective addition of water

“…39,[102][103][104][105][106] The substrate spectrum of malease isas is the case for fumaraserather narrow, and maleic acid and citraconic acid are accepted best, but small changes in the functional group pattern of these substrates are allowed. [104][105][106] For example chloromaleate and bromomaleate are hydrated to give α-substituted malates (2S,3S)-3-chloromalate and (2S,3S)-3-bromomalate. 107 In industry, Pseudomonas pseudoalcaligenes containing malease is used for the large-scale production of malic acid.…”

The selective addition of water to CC bonds; enzymes are the best chemists

“…Maleic acid is not a part of primary metabolism, nonetheless malease exists. 9,14,[42][43][44] This hydratase converts maleate into (R)-malate, thus complementing fumarase in its enantioselective catalytic activity. Similar to fumarase malease does not contain a cofactor and is relatively stable.…”

“…The purified enzyme weighs 89 kDa, consisting of two subunits with 57 and 24 kDa. 43 It also has a very limited substrate spectrum, accepting only maleic acid and citraconic acid. The catalytic addition of water converts both acids into the corresponding (R)-alcohols (Scheme 9).…”

“…[60][61][62] These catabolic routes have been applied industrially for the whole cell conversion of butyric acid via its thioester into Scheme 9 Malease and citraconase display similar stability and the same enantioselectivity in the conversion of their two substrates. 43,45 Scheme 10 Urocanase catalyses what at first glance seems to be a 1,6-addition of water. The mechanism, however, proceeds via a covalent NAD + adduct.…”