Construction of metal-organic framework-based multienzyme system for L-tert-leucine production.

Chiral compounds are important drug intermediates that play a critical role in human life. Herein, we report a facile method to prepare multi-enzyme nano-devices with high catalytic activity and stability. The self-assemble molecular binders SpyCatcher and SpyTag were fused with leucine dehydrogenase and glucose dehydrogenase to produce sc-LeuDH (SpyCatcher-fused leucine dehydrogenase) and GDH-st (SpyTag-fused glucose dehydrogenase), respectively. After assembling, the cross-linked enzymes LeuDH-GDH were formed. The crosslinking enzyme has good pH stability and temperature stability. The coenzyme cycle constant of LeuDH-GDH was always higher than that of free double enzymes. The yield of L-tert-leucine synthesis by LeuDH-GDH was 0.47 times higher than that by free LeuDH and GDH. To further improve the enzyme performance, the cross-linked LeuDH-GDH was immobilized on zeolite imidazolate framework-8 (ZIF-8) via bionic mineralization, forming LeuDH-GDH @ZIF-8. The created co-immobilized enzymes showed even better pH stability and temperature stability than the cross-linked enzymes, and LeuDH-GDH@ZIF-8 retains 70% relative conversion rate in the first four reuses. In addition, the yield of LeuDH-GDH@ZIF-8 was 0.62 times higher than that of LeuDH-GDH, and 1.38 times higher than that of free double enzyme system. This work provides a novel method for developing multi-enzyme nano-device, and the ease of operation of this method is appealing for the construction of other multi-enzymes @MOF systems for the applications in the kinds of complex environment.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Leucine Dehydrogenase: Structure and Thermostability.

Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD+-dependent oxidoreductase which catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH from Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has already been applied to quantify the concentration of BCAAs in biological specimens. However, the molecular mechanism of its thermostability had been unknown because no high-resolution structure was available. Here, we discuss the thermostability of GstLDH on the basis of its structure determined by cryo-electron microscopy. Sequence comparison with other structurally characterized LDHs (from Lysinibacillus sphaericus and Sporosarcina psychrophila) indicated that non-conserved residues in GstLDH, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric stability through intermolecular interactions between protomers. Furthermore, NAD+ binding to GstLDH increased the thermostability of the enzyme as additional intermolecular interactions formed on cofactor binding. This knowledge is important for further applications and development of amino acid metabolizing enzymes in industrial and clinical fields.

Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy.

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD+-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD+-bound LDH are reported at 3.0 and 3.2 Šresolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD+. NAD+ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD+-bound form was approximately three times higher than that of the apo form after incubation at 80 °C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.

Expression, purification and characterization of a thermostable leucine dehydrogenase from the halophilic thermophile Laceyella sacchari.

OBJECTIVE: A potential thermotolerant L-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized. RESULTS: Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C. CONCLUSION: It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.

Directed evolution of leucine dehydrogenase for improved efficiency of L-tert-leucine synthesis.

L-tert-Leucine and its derivatives are used as synthetic building blocks for pharmaceutical active ingredients, chiral auxiliaries, and ligands. Leucine dehydrogenase (LeuDH) is frequently used to prepare L-tert-leucine from the α-keto acid precursor trimethylpyruvate (TMP). In this study, a high-throughput screening method for the L-tert-leucine synthesis reaction based on a spectrophotometric approach was developed. Directed evolution strategy was applied to engineer LeuDH from Lysinibacillus sphaericus for improved efficiency of L-tert-leucine synthesis. After two rounds of random mutagenesis, the specific activity of LeuDH on the substrate TMP was enhanced by more than two-fold, compared with that of the wild-type enzyme, while the activity towards its natural substrate, leucine, decreased. The catalytic efficiencies (k cat/K m) of the best mutant enzyme, H6, on substrates TMP and NADH were all enhanced by more than five-fold as compared with that of the wild-type enzyme. The efficiency of L-tert-leucine synthesis by mutant H6 was significantly improved. A productivity of 1170 g/l/day was achieved for the mutant enzyme H6, compared with 666 g/l/day for the wild-type enzyme.

Construction of a reusable multi-enzyme supramolecular device via disulfide bond locking.

A strategy for constructing a reusable multi-enzyme supramolecular device was developed by reprogramming protein-protein interactions and disulfide bond locking. The resultant multi-enzyme supramolecular device demonstrated good reusability, and approximately 80% of its initial catalytic activity was retained even after eight cycles of reuse.

A novel chimeric amine dehydrogenase shows altered substrate specificity compared to its parent enzymes.

We created a novel chimeric amine dehydrogenase (AmDH) via domain shuffling of two parent AmDHs ('L- and F-AmDH'), which in turn had been generated from leucine and phenylalanine DH, respectively. Unlike the parent proteins, the chimeric AmDH ('cFL-AmDH') catalyzes the amination of acetophenone to (R)-methylbenzylamine and adamantylmethylketone to adamantylethylamine.

Amperometric bienzyme screen-printed biosensor for the determination of leucine.

Leucine plays an important role in protein synthesis, brain functions, building muscle mass, and helping the body when it undergoes stress. Here, we report a new amperometric bienzyme screen-printed biosensor for the determination of leucine, by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and leucine dehydrogenase (LDH) on a screen-printed electrode with NADP(+) and p-hydroxybenzoate as the cofactors. The detection principle of the sensor is that LDH catalyzes the specific dehydrogenation of leucine by using NADP(+) as a cofactor. The product, NADPH, triggers the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a change in electron concentration at the working carbon electrode, which is detected by the potentiostat. The sensor shows a linear detection range between 10 and 600 µM with a detection limit of 2 µM. The response is reproducible and has a fast measuring time of 5-10 s after the addition of a given concentration of leucine.

An enzymatic cyclopentyl[b]indole formation involved in scytonemin biosynthesis.

Previous studies of the biosynthetic enzymes involved in the assembly of scytonemin (1), a cyanobacterial sunscreen, have identified beta-ketoacid 2 as an important intermediate that is produced by ThDP-dependent enzyme ScyA. We now report that ScyC, previously annotated as a hypothetical protein, catalyzes cyclization and decarboxylation of 2 to generate ketone 5. Assembly of the cyclopentyl[b]indole structure in this manner has little precedent in the chemical literature. Additional mechanistic experiments have revealed that cyclization likely precedes decarboxylation and that the latter event may provide a driving force for cyclopentane formation.

A combinatorial approach to detect coevolved amino acid networks in protein families of variable divergence.

Communication between distant sites often defines the biological role of a protein: amino acid long-range interactions are as important in binding specificity, allosteric regulation and conformational change as residues directly contacting the substrate. The maintaining of functional and structural coupling of long-range interacting residues requires coevolution of these residues. Networks of interaction between coevolved residues can be reconstructed, and from the networks, one can possibly derive insights into functional mechanisms for the protein family. We propose a combinatorial method for mapping conserved networks of amino acid interactions in a protein which is based on the analysis of a set of aligned sequences, the associated distance tree and the combinatorics of its subtrees. The degree of coevolution of all pairs of coevolved residues is identified numerically, and networks are reconstructed with a dedicated clustering algorithm. The method drops the constraints on high sequence divergence limiting the range of applicability of the statistical approaches previously proposed. We apply the method to four protein families where we show an accurate detection of functional networks and the possibility to treat sets of protein sequences of variable divergence.

Cloning, protein sequence clarification, and substrate specificity of a leucine dehydrogenase from Bacillus sphaericus ATCC4525.

Although an X-ray model sequence of a leucine dehydrogenase from Bacillus sphaericus ATCC4525 was reported, the amino acid sequence of this enzyme has not been confirmed. In the current study, this leucine dehydrogenase gene was cloned, sequenced, and over-expressed in Escherichia coli, and the protein sequence has been clarified. This leucine dehydrogenase is not identical with that of B. sphaericus IFO3525 because there are 16 different amino acid residues between these two proteins. Since the information on the catalytic properties of leucine dehydrogenase from B. sphaericus ATCC4525 has been limited, the recombinant enzyme was purified as His-tagged protein and further studied. This enzyme showed activity toward aliphatic substrates for both oxidative deamination and reductive amination and is an effective catalyst for the asymmetric synthesis of alpha-amino acids from the corresponding alpha-ketoacids.