The enzyme, found in bacteria and fungi, can also accept a number of substituted mandelate derivatives, such as 3-hydroxymandelate, 4-hydroxymandelate, 2-methoxymandelate, 4-hydroxy-3-methoxymandelate and 3-hydroxy-4-methoxymandelate. The enzyme has no activity with (S)-mandelate (cf. EC 1.1.99.31, (S)-mandelate dehydrogenase) [1,2]. The enzyme transfers the pro-R-hydrogen from NADH .
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SYSTEMATIC NAME
IUBMB Comments
(R)-mandelate:NAD+ 2-oxidoreductase
The enzyme, found in bacteria and fungi, can also accept a number of substituted mandelate derivatives, such as 3-hydroxymandelate, 4-hydroxymandelate, 2-methoxymandelate, 4-hydroxy-3-methoxymandelate and 3-hydroxy-4-methoxymandelate. The enzyme has no activity with (S)-mandelate (cf. EC 1.1.99.31, (S)-mandelate dehydrogenase) [1,2]. The enzyme transfers the pro-R-hydrogen from NADH [2].
the N-terminal domain forms many more hydrogen bonds with NADH than the C-terminal domain in the binary complex, and moves together with the bound NADH molecule in the shear motion, these intermolecular hydrogen bonds are retained
neither stimulated nor inhibited by 1 mM concentrations of NaCl, Na2SO4, KCl, MgCl2, NH4Cl, CaCl2, ZnCl2, CoCl2, FeCl3, KH2PO4, NaH2PO4, MnSO4, MnCl2, or CuSO4
8 mM, greater than 95% inactivation after 2 h at 27°C. Inactivation is a pseudo-first-order process. D-Mandelate protects against inactivation. The inhibitor is covalently bound
neither stimulated nor inhibited by 1 mM concentrations of NaCl, Na2SO4, KCl, MgCl2, NH4Cl, CaCl2, ZnCl2, CoCl2, FeCl3, KH2PO4, NaH2PO4, MnSO4, MnCl2 or CuSO4. Not inhibited by the following metal-chelating agents each at 1 mM: EDTA, diethyldithiocarbamic acid, bathophenanthroline disulfonic acid, dihydroxybenzene disulfonic acid, pyrazole, 8-hydroxyquinoline or 2,2'-dipyridyl. Little or no inhibition of enzyme activity by 10 mM iodoacetate, 10 mM iodoacetamide, 6.65 mM 5,5'-dithiobis(2-nitrobenzoic acid) or 5 mM 4-chloromercuribenzoate, even after a 6 h preincubation at 27°C. Neither inhibited nor stimulated by 1 mM concentrations of benzaldehyde, benzoate, succinate, glucose, ATP/Mg2+, ADP/Mg2+ or acetyl-CoA
enzyme D-ManDH2 forms many hydrogen bonds with the 2-ketoacid moiety of AOA in the ternary complex structure, involving Asn105, Lys187, Asn191, Asn195 and Ser260, substrate binding structures in binary and tertiary complexes, structure and structure-function analysis, overview. The substrate binding induces a shear motion of the N-terminal domain along the C-terminal domain, following the hinge motion induced by the NADH binding, and allows the bound NADH molecule to form favorable interactions with a 2-oxoacid substrate. D-ManDH possesses a sufficiently wide pocket that accommodates the C3 branched side chains of substrates like KPR, but unlike the pocket of KPR, the pocket of D-ManDH comprises an entirely hydrophobic surface and an expanded space, in which the AOA benzene is accommodated. The expanded space mostly comprises a mobile loop structure, which likely modulates the shape and size of the space depending on the substrate. D-ManDH2 undergoes the opening and closing motion in the entrance area of the substrate binding pocket, which comprises mostly Met128, Thr130 and Ile254, between the binary and ternary complexes. Lys187 is actually the acid/base catalyst of the enzyme
D-ManDH2 constitutively forms a dimeric structure. Each of the two subunits binds the NADH and AOA molecules between the N-terminal (residues 1-176) and C-terminal (residues 180-311). The intersubunit contact is formed between the two C-terminal domains of the dimer on the opposite side of NADH and AOA, and exhibits no significant structural change among the three D-ManDH2 structures. The N-terminal domain exhibits a shear motion along the C-terminal domain between the binary and ternary complex structures, following the domain closure through the hinge motion between the apo and binary complex structures. Residue Asn105 forms hydrogen bonds with both the nicotinamide-ribose of NADH and the carbonyl oxygen of AOA in the ternary complex structure, suggesting that this residue promotes the shear motion of the N-terminal domain
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified enzyme in complex with substrates NADH and anilino(oxo)acetate (AOA), hanging-drop vapor diffusion method, mixing of 10 mg/ml protein in 5 mM sodium MOPS, pH 7.5, 2 mM NADH, and 20 mM AOA with reservoir solution containing 0.1 M cacodylate-Na, pH 5.0, 0.17 M ammonium sulfate, and 22.5% PEG 8000, in a 1:1 ratio, at 25°C, X-ray diffraction structure determination and analysis at 2.4 A resolution, molecular replacement using the crystal structure of unliganded D-ManDH2 (PDB ID 3WFI) as a search model, modeling. The ternary complex of D-ManDH2 forms a 2fold symmetric homodimer in a crystallographic asymmetric unit, as in the cases of the apo and binary complex structures
the enzyme is crystallized in three different forms using the hanging drop vapour diffusion method at 15-20°C. Type I crystals belong to space group P222(1), P22(1)2(1) or P2(1)2(1)2(1) with a = 100.3 A, b = 117.4 A, c = 80.4 A and are likely to contain a dimer in the crystallographic asymmetric unit
by coculturing two Escherichia coli strains expressing LhDMDH and lactate dehydrogenase (LDH) from Lactobacillus casei, LcLDH, a system for the efficient synthesis of phenylglyoxylic acid (PGA) is developed, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA. Whole cell catalysis is a preferred option to eliminate the influence of low thermostability of enzyme LhDMDH in the biosynthesis of PGA
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene cloning, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21, coexpression with LDH from Lactobacillus casei increasing the phenylglyoxylate yield
D-mandelate dehydrogenase (DMDH) has the potential to convert D-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. Development of an alternative strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA