Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant <i>meso</i>‐Diaminopimelate Dehydrogenase

Liu, Weidong; Guo, Rey‐Ting; Chen, Xi; Li, Zhe; Gao, Xuemin; Huang, Chun‐Hsiang; Wu, Qingping; Feng, Jinhui; Zhu, Dunming

doi:10.1002/cbic.201402632

Cited by 19 publications

(21 citation statements)

References 28 publications

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“…DapDH catalyzes the biosynthesis of meso -DAP, which can be used as processor for the biosynthesis of peptidoglycan and L-lysine (Fig. 1) 13 . However, the excessive increase in cell growth is not good for L-lysine production because more carbon source enter into the biosynthesis of peptidoglycan rather than L-lysine.…”

Section: Resultsmentioning

confidence: 99%

“…The dehydrogenase pathway converts THDPA to meso -DAP in a single step, which is catalyzed by diaminopimelate dehydrogenase (DapDH; encoded by ddh gene) 13 . However, the dehydrogenase pathway is only found in a handful of species of bacteria, which is in contrast to the alternative succinylase and acetylase pathways that are the most widely distributed in plants and bacteria 14 .…”

Section: Introductionmentioning

confidence: 99%

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Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli

Ruan

Liu

et al. 2019

Sci Rep

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Dehydrogenase pathway, one of diaminopimelate pathway, is important to the biosynthesis of L-lysine and peptidoglycan via one single reaction catalyzed by meso-diaminopimelate dehydrogenase (DapDH). In this study, the thermostable DapDH was introduced into diaminopimelate pathway that increased the final titer (from 71.8 to 119.5 g/L), carbon yield (from 35.3% to 49.1%) and productivity (from 1.80 to 2.99 g/(L∙h)) of L-lysine by LATR12-2∆rpiB::ddhSt in fed-batch fermentation. To do this, the kinetic properties and the effects of different DapDHs on L-lysine production were investigated, and the results indicated that overexpression of StDapDH in LATR12-2 was beneficial to construct an L-lysine producer with good productive performance because it exhibited the best of kinetic characteristics and optimal temperature as well as thermostability in reductive amination. Furthermore, ammonium availability was optimized, and found that 20 g/L of (NH4)2SO4 was the optimal ammonium concentration for improving the efficiency of L-lysine production by LATR12-2∆rpiB::ddhSt. Metabolomics analysis showed that introducing the StDapDH significantly enhanced carbon flux into pentose phosphate pathway and L-lysine biosynthetic pathway, thus increasing the levels of NADPH and precursors for L-lysine biosynthesis. This is the first report of a rational modification of diaminopimelate pathway that improves the efficiency of L-lysine production through overexpression of thermostable DapDH in E. coli.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli

Ruan

Liu

et al. 2019

Sci Rep

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“…KACA (yellow) and NADPH (magenta) molecules are shown as stick models. The final A -weighted ( Because DAPDH acts stereoselectively on the D-center of meso-DAP, studies of DAPDH mutations aimed at changing its substrate spectrum have so far been focused on the amino acid residues around the L-amino acid center of the substrate (7,13). To enlarge the substrate-binding pocket of S. thermophilum DAPDH, for example, the four residues (Phe146, Thr171, Arg181, and His227) interacting with the L-center were chosen for site saturation mutagenesis (7).…”

Section: Resultsmentioning

confidence: 99%

“…We have also determined the structures of U. thermosphaericus DAPDH in the apo form and in complex with NADP ϩ and Ntris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) (12). Furthermore, the structures of a Clostridium tetani DAPDH mutant exhibiting catalytic ability for the synthesis of D-amino acids (e.g., D-alanine and D-phenylalanine) have been reported (13). Extensive analysis of these structures has shed light on the structure of the substrate-binding site, the structural features responsible for the high thermostability of thermophilic DAPDHs, and the factors underlying the change in substrate recognition between DAPDH and DAADH caused by introducing mutations.…”

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confidence: 99%

Structure-Based Engineering of an Artificially Generated NADP ⁺ -Dependent d -Amino Acid Dehydrogenase

Hayashi

Seto

Akita

et al. 2017

Appl Environ Microbiol

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A stable NADP-dependent d-amino acid dehydrogenase (DAADH) was recently created from -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

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“…This S. thermophilum thermostable meso -DAPDH has been reported to exhibit somewhat unusual substrate specificity compared with those of C. glutamicum and U. thermosphaericus counterparts. After that, structural and mutational studies on S. thermophilum and Clostridium tetani meso -DAPDHs followed to obtain new types of D -AADH ( Liu et al, 2014 , 2015 ). In this review, the creation of novel thermostable D -AADHs and their useful application are described focusing on our recent research results.…”

Section: Introductionmentioning

confidence: 99%

Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

et al. 2018

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Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.

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Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

Cited by 19 publications

References 28 publications

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli

Structure-Based Engineering of an Artificially Generated NADP ⁺ -Dependent d -Amino Acid Dehydrogenase

Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

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Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

Cited by 19 publications

References 28 publications

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli

Overexpression of thermostable meso-diaminopimelate dehydrogenase to redirect diaminopimelate pathway for increasing L-lysine production in Escherichia coli

Structure-Based Engineering of an Artificially Generated NADP + -Dependent d -Amino Acid Dehydrogenase

Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

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Product

Resources

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Structure-Based Engineering of an Artificially Generated NADP ⁺ -Dependent d -Amino Acid Dehydrogenase