the enzyme barely shows any activity towards (S)-beta-aminobutyric acid, beta-alanine, (R)-beta-homoserine, (R)-beta-homomethionine, (S)-beta-homoglutamine, (R)-beta-phenylalanine, and (S)-beta-homolysine
either NAD+ or NADP+ serve as coenzyme. Double-reciprocal plots of activity versus pyridine nucleotide concentration are biphasic. The Km values for NADP+ are lower but a higher Vmax is observed with NAD+
either NAD+ or NADP+ serve as coenzyme. Double-reciprocal plots of activity versus pyridine nucleotide concentration are biphasic. The Km values for NADP+ are lower but a higher Vmax is observed with NAD+
either NAD+ or NADP+ serve as coenzyme. Double-reciprocal plots of activity versus pyridine nucleotide concentration are biphasic. The Km values for NADP+ are lower but a higher Vmax is observed with NAD+
either NAD+ or NADP+ serve as coenzyme. Double-reciprocal plots of activity versus pyridine nucleotide concentration are biphasic. The Km values for NADP+ are lower but a higher Vmax is observed with NAD+
at pH 8.9 Mn2+ gives the highest increase in activity (232% at 0.5 mM). Concentrations of 0.01, 0.05, 0.1, 0.75, and 1.0 mM increase the enzyme activity by 110, 156, 188, 258, and 263%, respectively. The activity is maximal at 0.75 mM Mn2+
addition of any of the neutral salts causes a parabolic inhibition. A direct comparison of arsenate and chloride ion shows that arsenate is not as inhibitory even though it has a higher ionic strength. Arsenate interacts with the enzyme differently from chloride and bromide. Sulfate, which is a large ion like arsenate, is as inhibitory as chloride (at equivalent ionic strength) with NAD+ as coenzyme, but it is much less effective than chloride with NADP+ as coenzyme
addition of any of the neutral salts causes a parabolic inhibition. A direct comparison of arsenate and chloride ion shows that arsenate is not as inhibitory even though it has a higher ionic strength. Arsenate interacts with the enzyme differently from chloride and bromide. Sulfate, which is a large ion like arsenate, is as inhibitory as chloride (at equivalent ionic strength) with NAD+ as coenzyme, but it is much less effective than chloride with NADP+ as coenzyme
addition of any of the neutral salts causes a parabolic inhibition. A direct comparison of arsenate and chloride ion shows that arsenate is not as inhibitory even though it has a higher ionic strength. Arsenate interacts with the enzyme differently from chloride and bromide. Sulfate, which is a large ion like arsenate, is as inhibitory as chloride (at equivalent ionic strength) with NAD+ as coenzyme, but it is much less effective than chloride with NADP+ as coenzyme
addition of any of the neutral salts causes a parabolic inhibition. A direct comparison of arsenate and chloride ion shows that arsenate is not as inhibitory even though it has a higher ionic strength. Arsenate interacts with the enzyme differently from chloride and bromide. Sulfate, which is a large ion like arsenate, is as inhibitory as chloride (at equivalent ionic strength) with NAD+ as coenzyme, but it is much less effective than chloride with NADP+ as coenzyme
the lactams of DL-erythro-3,5-diaminohexanoate and DL-threo-3,5-diaminohexanoatae and 2-methylpyrrolidone-5-carboxylic acid have no effect on activity. Acetate (10 mM), butyrate (10 mM), acetyl phosphate (2 mM), or acetyl-CoA (0.62 mM) have no effect on enzyme activity
in the proposed mechanism, the catalytic cycle starts with the hydride transfer from C3 of L-erythro-3,5-diaminohexanoate to C4 of NADP+ with a barrier of 18.8 kcal/mol. The generated iminium intermediate (Int1) is 1.8 kcal/mol lower than the ternary complex E:DAH. In the next hydration step, both the deprotonated C5-amino group of L-erythro-3,5-diaminohexanoate and D177 can be the catalytic base to activate the water molecule that attacks the electrophilic carbon of Int1. The barrier is 8.3 kcal/mol or 12.4 kcal/mol relative to Int1 for the pathways with C5-amino group or D177, respectively. For the former pathway, after the formation of hydrated intermediate (Int2), a proton transfer takes place from the protonated C5-amino group to D177 via the newly formed hydroxyl group with a barrier of 10.7 kcal/mol relative to Int1, arriving at the same intermediate (Int3) as the latter pathway. Then, a proton transfer from D177 to C3-amino group results in intermediate Int4 with -0.1 kcal/mol energy, in which the protonated amino group can be significantly stabilized by the carboxylate groups of D49 and D177. Finally, the products are formed by the C-N bond cleavage, concurrently with intramolecular proton transfer from the C3-hydroxyl group to the C5-amine
in the proposed mechanism, the catalytic cycle starts with the hydride transfer from C3 of L-erythro-3,5-diaminohexanoate to C4 of NADP+ with a barrier of 18.8 kcal/mol. The generated iminium intermediate (Int1) is 1.8 kcal/mol lower than the ternary complex E:DAH. In the next hydration step, both the deprotonated C5-amino group of L-erythro-3,5-diaminohexanoate and D177 can be the catalytic base to activate the water molecule that attacks the electrophilic carbon of Int1. The barrier is 8.3 kcal/mol or 12.4 kcal/mol relative to Int1 for the pathways with C5-amino group or D177, respectively. For the former pathway, after the formation of hydrated intermediate (Int2), a proton transfer takes place from the protonated C5-amino group to D177 via the newly formed hydroxyl group with a barrier of 10.7 kcal/mol relative to Int1, arriving at the same intermediate (Int3) as the latter pathway. Then, a proton transfer from D177 to C3-amino group results in intermediate Int4 with -0.1 kcal/mol energy, in which the protonated amino group can be significantly stabilized by the carboxylate groups of D49 and D177. Finally, the products are formed by the C-N bond cleavage, concurrently with intramolecular proton transfer from the C3-hydroxyl group to the C5-amine
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
structures of 3,5-DAHDH in apo form, complexed with cofactor NADPH, and of variant E310G/A314Y in complex with NADPH. The overall monomeric structure of 3,5-DAHDH is composed of an N-terminal catalytic domain (Domain I) and a C-terminal cofactor-binding domain (Domain II) separating by a deep cleft
mutant exhibited an about 200 times enhanced activity towards substrate (R)-beta-homomethionine compared to mutant E310G and increased activities with (S)-beta-homolysine, (S)-beta-aminobutyric acid, (R)-beta-phenylalanine, (S)-beta-homophenylalanine
the mutant exhibits activity towards (S)-beta-aminobutyric acid, beta-alanine, (R)-beta-homoserine, (R)-beta-homomethionine, (S)-beta-homoglutamine, (R)-beta-phenylalanine, and (S)-beta-homolysine
the mutant exhibits activity towards (S)-beta-aminobutyric acid, beta-alanine, (R)-beta-homoserine, (R)-beta-homomethionine, (S)-beta-homoglutamine, (R)-beta-phenylalanine, and (S)-beta-homolysine
the mutant exhibits activity towards (S)-beta-aminobutyric acid, beta-alanine, (R)-beta-homoserine, (R)-beta-homomethionine, (S)-beta-homoglutamine, (R)-beta-phenylalanine, and (S)-beta-homolysine
the mutant exhibits activity towards (S)-beta-aminobutyric acid, beta-alanine, (R)-beta-homoserine, (R)-beta-homomethionine, (S)-beta-homoglutamine, (R)-beta-phenylalanine, and (S)-beta-homolysine
mutation E310G destroys the hydrogen bond interaction with the amide group of NADPH observed in the wild-type structure. Mutant shows improved activity towards substrate (R)-beta-homomethionine
t1/2: 32 min, inactivation follows first-order kinetics. In the presence of both NAD+ and 3,5-diaminohexanoate, the enzyme is effectively stabilized against heat inactivation at 55°C. NAD+ alone accelerates heat inactivation
Crystal structures and catalytic mechanism of L-erythro-3,5-diaminohexanoate dehydrogenase and rational engineering for asymmetric synthesis of beta-amino acids