Structural Insights into l -Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme

Abstract

L-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (Ͼ30%)/homology (Ͼ50%) with NAD(P) ϩ -dependent L-Glu/L-Leu/L-Phe/L-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of L-Trp to 3-indolepyruvate in the presence of NAD ϩ . Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of L-Glu/L-Leu/L-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD ϩ /NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the ␤-1 strand of each subunit, as was observed in the case of L-Phe dehydrogenase. The binding site for the substrate L-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate L-Trp, were arranged similarly to that found in L-Leu/L-Phe dehydrogenases but fairly different from that of an L-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, L-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme.IMPORTANCE In this study, we determined the three-dimensional structure of L-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any L-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate L-Trp. These studies show the structural characteristics of L-Trp dehydrogenase and hence would facilitate novel applications of L-Trp dehydrogenase.

The Symmetric Active Site of Enantiospecific Enzymes

“…In AAOs, the reduced cofactor (flavin adenine mono or dinucleotide, FMNH 2 or FADH 2, respectively) is reoxidized by a molecular oxygen molecule producing hydrogen peroxide (Figure 2A,B). On the other hand, in AADHs, the electrons deriving from the amino acid oxidation are transferred through the reduced cofactors FADH 2 or nicotinamide adenine dinucleotide (phosphate), NAD(P)H, to a membrane-associated electron acceptor (usually a molecule belonging to the coenzyme Q family) [29,30]. This electron transfer/reoxidation system allows AADHs to perform the oxidative deamination of amino acids even under anaerobic conditions.…”

Enhanced Catalytic Efficiency of L‐amino Acid Deaminase Achieved by a Shorter Hydride Transfer Distance

“…Currently, enzymatic synthesis of α-keto acids relies on the use of amino acid transaminase (AAT, EC 2.1.1.X), [11] L-amino acid dehydrogenase (LAADH, EC 1.4.1.5), [10a,12] amino acid oxidase (DAAO, EC 1.1.3.3; LAAO, EC 1.4.3.3) [13] (Scheme S1), and L-amino acid deaminase (LAAD, EC 1.4.3.2) [4,10b,c,14] (Scheme 1). However, all of these enzymes suffer from considerable limitations: AAT and LAADH present low conversion rates due to simultaneous reversible reactions; AAT-catalyzed transamination requires additional amino acceptors (e. g., α-ketoglutaric acid); [11a] LAADH requires NAD, a costly compound, as a cofactor, [12] as well as complex separation and purification of the products; and LAAO-catalyzed deamination releases toxic byproducts (e. g., H 2 O 2 ) and the difficulties in its recombinant production; [13b] DAAT and DAAO can also produce keto acids, but the substrates used by the two enzymes are unnatural Dtype amino acids which are more expensive than L-type amino acids. Therefore, these enzymes may not be suitable for the industrial production of α-keto acids.…”

Enhanced catalytic efficiency and universality of L-amino acid deaminase achieved by a shorter proton transfer distance

“…Currently, enzymatic synthesis of α-keto acids relies on the use of amino acid transaminase (AAT, EC 2.1.1.X) (Taylor et al 1998;Wuet al 2018), L-amino acid dehydrogenase (LAADH, EC 1.4.1.5) (Jambunathan et al 2014;Wakamatsu et al 2017), amino acid oxidase (DAAO,EC 1.1.3.3;LAAO,EC 1.4.3.3) (Asano et al 2019;Hossain et al 2014a;Mattevi et al 1996;Nakano et al 2019) , and Lamino acid deaminase (LAAD, EC 1.4.3.2) (Ju et al 2017;Ju et al 2016;Liu et al2013;Molla et al 2017;Motta et al 2016;Pei et al 2020;Song et al 2016;Wang et al 2019;Wu et al 2020) (Schemes 1 and 2). However, all of these enzymes suffer from considerable limitations: AAT and LAADH present low conversion rates due to simultaneous reversible reactions; AAT-catalyzed transamination requires additional amino acceptors (e.g., α-ketoglutaric acid) (Tayloret al 1998); LAADH requires NAD, a costly compound, as a cofactor (Wakamatsu et al 2017), as well as complex separation and purification of the products; and LAAO-catalyzed deamination releases toxic byproducts (e.g., H 2 O 2 ) and the difficulties in its recombinant production (Hossain et al2014a); DAAT and DAAO can also produce keto acids, but the substrates involved are expensive unnatural amino acids. Therefore, these enzymes are not suitable for the industrial production of α-keto acids.…”