Cloning and expression heterologous alanine dehydrogenase genes: Investigation of reductive amination potential of L-alanine dehydrogenases for green synthesis of alanine derivatives.

Unnatural amino acids (UAAs) offer significant promise in a wide range of applications, including drug discovery, the custom design of peptides and proteins, and their utility and use as markers for monitoring molecular interactions in biological research. The synthesis of UAAs presents a formidable challenge and can be classified into two primary categories: enzymatic and chemical synthesis. Notably, the enzymatic route, specifically asymmetric synthesis, emerges as a an attractive method for procuring enantiopure UAAs with high efficiency, owing to its streamlined and concise reaction mechanism. The current study investigated the reductive amination activity mechanisms of alanine dehydrogenase (L-AlaDH), sourced from a combination of newly and previously characterized microorganisms. Our principal aim was to evaluate the catalytic efficiency of these L-AlaDH enzymes concerning a range of specific ketoacids and pyruvate to ascertain their capability for facilitating the production of both natural and unnatural amino acids. After the characterization processes, mutation points for TtAlaDH were determined and as a result of the mutations, mutants that could use ketocaproate and ketovalerate more effectively than the wild type were obtained. Among the enzymes studied, MetAlaDH exhibited the highest specific activity against pyruvate, 173 U/mg, and a KM value of 1.3 mM. VlAlaDH displayed the most favourable catalytic efficiency with a rate constant of 170 s-1mM-1. On the other hand, AfAlaDH demonstrated the highest catalytic efficiency against α-ketobutyrate (34.0 s-1mM-1) and α-ketovalerate (2.7 s-1mM-1). Of the enzymes investigated in the study, TtAlaDH exhibited the highest effectiveness among bacterial enzymes in catalyzing ketocaproate with a measured catalytic efficiency of about 0.6 s-1mM-1 and a KM value of approximately 0.3 mM. These findings provide valuable insights into the substrate specificity and catalytic performance of L-AlaDHs, enhancing our understanding of their potential applications in various biocatalytic processes.

Alanine dehydrogenases from four different microorganisms: characterization and their application in L-alanine production.

BACKGROUND: Alanine dehydrogenase (AlaDH) belongs to oxidoreductases, and it exists in several different bacteria species and plays a key role in microbial carbon and nitrogen metabolism, spore formation and photosynthesis. In addition, AlaDH can also be applied in biosynthesis of L-alanine from cheap carbon source, such as glucose. RESULTS: To achieve a better performance of L-alanine accumulation, system evaluation and comparison of different AlaDH with potential application value are essential. In this study, enzymatic properties of AlaDH from Bacillus subtilis 168 (BsAlaDH), Bacillus cereus (BcAlaDH), Mycobacterium smegmatis MC2 155 (MsAlaDH) and Geobacillus stearothermophilus (GsAlaDH) were firstly carefully investigated. Four different AlaDHs have few similarities in optimum temperature and optimum pH, while they also exhibited significant differences in enzyme activity, substrate affinity and enzymatic reaction rate. The wild E. coli BL21 with these four AlaDHs could produce 7.19 g/L, 7.81 g/L, 6.39 g/L and 6.52 g/L of L-alanine from 20 g/L glucose, respectively. To further increase the L-alanine titer, competitive pathways for L-alanine synthesis were completely blocked in E. coli. The final strain M-6 could produce 80.46 g/L of L-alanine with a yield of 1.02 g/g glucose after 63 h fed-batch fermentation, representing the highest yield for microbial L-alanine production. CONCLUSIONS: Enzyme assay, biochemical characterization and structure analysis of BsAlaDH, BcAlaDH, MsAlaDH and GsAlaDH were carried out. In addition, application potential of these four AlaDHs in L-alanine productions were explored. The strategies here can be applied for developing L-alanine producing strains with high titers.

Efficient production of hydroxymethyl-2-furfurylamine by chemoenzymatic cascade catalysis of bread waste in a sustainable approach.

In this study, efficient and sustainable conversion of waste bread (WB) to 5-hydroxymethyl-2-furoamine (HMFA) was achieved in a cascade reaction in betaine:malonic acid (B:MA) - water. 5-HMF (30.3 wt% yield) was synthesized from WB (40.0 g/L) in B:MA - water (B:MA, 18 wt%) in 45 min at 190 °C. By using the newly created recombinant E. coli HNILGD-AlaDH cells expressing L-alanine dehydrogenase (AlaDH) and ω-transaminase mutant HNILGD as biocatalyst, the WB-valorized 5-HMF was biologically aminated into HMFA in a high yield (92.1%) at 35 °C for 12 h through in situ removal of the amino transfer by-products of the amine donor, greatly reducing amine donor dosage (from D-Ala/5-HMF = 16/1 to D-Ala/5-HMF = 2/1, mol/mol) and improving the productivity of HMFA (0.282 g HMFA per g WB). This two-step chemical-enzymatic cascade reaction strategy with B:MA and HNILGD-AlaDH whole-cell provides a new idea for the chemoenzymatic synthesis of valuable furan chemicals from waste biomass.

Application of reductive amination by heterologously expressed Thermomicrobium roseumL-alanine dehydrogenase to synthesize L-alanine derivatives.

Unnatural amino acids are unique building blocks in modern medicinal chemistry as they contain an amino and a carboxylic acid functional group, and a variable side chain. Synthesis of pure unnatural amino acids can be made through chemical modification of natural amino acids or by employing enzymes that can lead to novel molecules used in the manufacture of various pharmaceuticals. The NAD+ -dependent alanine dehydrogenase (AlaDH) enzyme catalyzes the conversion of pyruvate to L-alanine by transferring ammonium in a reversible reductive amination activity. Although AlaDH enzymes have been widely studied in terms of oxidative deamination activity, reductive amination activity studies have been limited to the use of pyruvate as a substrate. The reductive amination potential of heterologously expressed, highly pure Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) was examined with regard to pyruvate, α-ketobutyrate, α-ketovalerate and α-ketocaproate. The biochemical properties were studied, which included the effects of 11 metal ions on enzymatic activity for both reactions. The enzyme accepted both derivatives of L-alanine (in oxidative deamination) and pyruvate (in reductive amination) as substrates. While the kinetic KM values associated with the pyruvate derivatives were similar to pyruvate values, the kinetic kcat values were significantly affected by the side chain increase. In contrast, KM values associated with the derivatives of L-alanine (L-α-aminobutyrate, L-norvaline, and L-norleucine) were approximately two orders of magnitude greater, which would indicate that they bind very poorly in a reactive way to the active site. The modeled enzyme structure revealed differences in the molecular orientation between L-alanine/pyruvate and L-norleucine/α-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.

Construction of an alanine dehydrogenase gene deletion strain for vaccine development against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂).

Nocardia seriolae is the main pathogen of fish nocardiosis. In our previous study, alanine dehydrogenase was identified as a potential virulence factor of N. seriolae. On the basis of this fact, the alanine dehydrogenase gene of N. seriolae (NsAld) was knocked out to establish the strain ΔNsAld for vaccine development against fish nocardiosis in this study. The LD50 of strain ΔNsAld was 3.90 × 105 CFU/fish, higher than that of wild strain (5.28 × 104 CFU/fish) significantly (p < 0.05). When the strain ΔNsAld was used as a live vaccine to immunize hybrid snakehead (Channa maculata ♀ × Channa argus ♂) at 2.47 × 105 CFU/fish by intraperitoneal injection, the non-specific immune indexes (LZM, CAT, AKP, ACP and SOD activities), specific antibody (IgM) titers and several immune-related genes (CD4, CD8α, IL-1ß, MHCIα, MHCIIα and TNFα) were up-regulated in different tissues, indicating that this vaccine could induce humoral and cell-mediated immune responses. Furthermore, the relative percentage survival (RPS) of ΔNsAld vaccine was calculated as 76.48% after wild N. seriolae challenge. All these results suggest that the strain ΔNsAld could be a potential candidate for live vaccine development to control fish nocardiosis in aquaculture.

Two different alanine dehydrogenases from Geobacillus kaustophilus: Their biochemical characteristics and differential expression in vegetative cells and spores.

Two putative alanine dehydrogenase (AlaDH) genes (GK2752 and GK3448) were found in the genome of a thermophilic spore-forming bacterium, Geobacillus kaustophilus. The amino acid sequences deduced from the two genes showed mutually high homology (71%), and the phylogenetic tree based on the amino acid sequences of the two putative AlaDHs and the homologous proteins showed that the two putative AlaDH genes (GK2752 and GK3448) belong to different groups. Both of the recombinant gene products exhibited high NAD+-dependent AlaDH activity and were purified to homogeneity and characterized in detail. Both enzymes showed high stability against low and high pHs and high temperatures (70 °C). Kinetic analyses showed that the activities of both enzymes proceeded according to the same sequentially ordered Bi-Ter mechanism. X-ray crystallographic analysis showed the two AlaDHs to have similar homohexameric structures. Notably, GK3448-AlaDH was detected in vegetative cells of G. kaustophilus but not spores, while GK2752-AlaDH was present only in the spores. This is the first report showing the presence of two AlaDHs separately expressed in vegetative cells and spores.

Alanine synthesized by alanine dehydrogenase enables ammonium-tolerant nitrogen fixation in Paenibacillus sabinae T27.

Most diazotrophs fix nitrogen only under nitrogen-limiting conditions, for example, in the presence of relatively low concentrations of NH4+ (0 to 2 mM). However, Paenibacillus sabinae T27 exhibits an unusual pattern of nitrogen regulation of nitrogen fixation, since although nitrogenase activities are high under nitrogen-limiting conditions (0 to 3 mM NH4+) and are repressed under conditions of nitrogen sufficiency (4 to 30 mM NH4+), nitrogenase activity is reestablished when very high levels of NH4+ (30 to 300 mM) are present in the medium. To further understand this pattern of nitrogen fixation regulation, we carried out transcriptome analyses of P. sabinae T27 in response to increasing ammonium concentrations. As anticipated, the nif genes were highly expressed, either in the absence of fixed nitrogen or in the presence of a high concentration of NH4+ (100 mM), but were subject to negative feedback regulation at an intermediate concentration of NH4+ (10 mM). Among the differentially expressed genes, ald1, encoding alanine dehydrogenase (ADH1), was highly expressed in the presence of a high level of NH4+ (100 mM). Mutation and complementation experiments revealed that ald1 is required for nitrogen fixation at high ammonium concentrations. We demonstrate that alanine, synthesized by ADH1 from pyruvate and NH4+, inhibits GS activity, leading to a low intracellular glutamine concentration that prevents feedback inhibition of GS and mimics nitrogen limitation, enabling activation of nif transcription by the nitrogen-responsive regulator GlnR in the presence of high levels of extracellular ammonium.

Antibacterial Activity of Squaric Amide Derivative SA2 against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA)-caused infection is difficult to treat because of its resistance to commonly used antibiotic, and poses a significant threat to public health. To develop new anti-bacterial agents to combat MRSA-induced infections, we synthesized novel squaric amide derivatives and evaluated their anti-bacterial activity by determining the minimum inhibitory concentration (MIC). Additionally, inhibitory activity of squaric amide 2 (SA2) was measured using the growth curve assay, time-kill assay, and an MRSA-induced skin infection animal model. A scanning electron microscope and transmission electron microscope were utilized to observe the effect of SA2 on the morphologies of MRSA. Transcriptome analysis and real-time PCR were used to test the possible anti-bacterial mechanism of SA2. The results showed that SA2 exerted bactericidal activity against a number of MRSA strains with an MIC at 4-8 µg/mL. It also inhibited the bacterial growth curve of MRSA strains in a dose-dependent manner, and reduced the colony formation unit in 4× MIC within 4-8 h. The infective lesion size and the bacterial number in the MRSA-induced infection tissue of mice were reduced significantly within 7 days after SA2 treatment. Moreover, SA2 disrupted the bacterial membrane and alanine dehydrogenase-dependent NAD+/NADH homeostasis. Our data indicates that SA2 is a possible lead compound for the development of new anti-bacterial agents against MRSA infection.

Fusion of Formate Dehydrogenase and Alanine Dehydrogenase as an Amino Donor Regenerating System Coupled to Transaminases.

Fusion enzymes are attractive tools for facilitating the assembly of biocatalytic cascades for chemical synthesis. This approach can offer great advantages for cooperative redox cascades that need the constant supply of a donor molecule. In this work, we have developed a self-sufficient bifunctional enzyme that can be coupled to transaminase-catalyzed reactions for the efficient recycling of the amino donor (L-alanine). By genetic fusion of an alanine dehydrogenase (AlaDH) and a formate dehydrogenase (FDH), a redox-complementary system was applied to recycle the amino donor and the cofactor (NADH), respectively. AlaDH and FDH were assembled in both combinations (FDH-AlaDH and AlaDH-FDH), with a 2.5-fold higher enzymatic activity of the latter system. Then, AlaDH-FDH was coupled to two different S-selective transaminases for the synthesis of vanillyl amine (10 mM) reaching up to 99 % conversion in 24 h in both cases. Finally, the multienzyme system was reused for at least 3 consecutive cycles when implemented in dialysis-assisted biotransformations.

Enzymatic determination of d-alanine with l-alanine dehydrogenase and alanine racemase.

An enzymatic assay system of d-Ala, which is reported to affect the taste, was constructed using alanine racemase and l-alanine dehydrogenase. d-Ala is converted to l-Ala by alanine racemase and then deaminated by l-alanine dehydrogenase with the reduction of NAD+ to NADH, which is determined with water-soluble tetrazolium. Using the assay system, the d-Ala contents of 7 crustaceans were determined.

Heterologous Expression and Partial Characterization of a New Alanine Dehydrogenase from Amycolatopsis sulphurea.

A novel alanine dehydrogenase (AlaDH; EC.1.4.1.1) was isolated from Amycolatopsis sulphurea and the AlaDH gene was cloned into a pET28a(+) plasmid and expressed in E. coli BL21 (DE3). The molecular mass of this enzyme was calculated as 41.09 kDa and the amino acid residues of the pure protein indicated the presence of N terminus polyhistidine tags. Its enzyme kinetic values were Km 2.03 mM, kcat 13.24 (s-1), and kcat/Km 6.53 (s-1 mM-1). AlaDH catalyzes the reversible conversion of L-alanine and pyruvate, which has an important role in the TCA energy cycle. Maximum AlaDH activity occurred at about pH 10.5 and 25 °C for the oxidative deamination of L-alanine. AlaDH retained about 10% of its relative activity at 55 °C and it remained about 90% active at 50 °C. These findings show that the AsAlaDH from A. sulphurea has the ability to produce valuable molecules for various industrial purposes and could represent a new potential biocatalyst for biotechnological applications after further characterization and improvement of its catalytic properties.

Function and Characterization of an Alanine Dehydrogenase Homolog From Nocardia seriolae.

Fish nocardiosis is a chronic, systemic, granulomatous disease in aquaculture. Nocardia seriolae has been reported to be one of the main pathogenic bacteria of fish nocardiosis. There are few studies on the associated virulence factors and pathogenesis of N. seriolae. Alanine dehydrogenase (ALD), which may be a secreted protein, was discovered by analysis using bioinformatics methods throughout the whole genomic sequence of N. seriolae. Nevertheless, the roles of ALD and its homologs in the pathogenesis of N. seriolae are not demonstrated. In this study, the function of N. seriolae ALD (NsALD) was preliminarily investigated by gene cloning, host cell subcellular localization, secreted protein identification, and cell apoptosis detection. Identification of the extracellular products of N. seriolae via mass spectrometry (MS) analysis revealed that NsALD is a secreted protein. In addition, subcellular localization of NsALD-GFP recombinant protein in fathead minnow (FHM) cells showed that the strong green fluorescence co-localized with the mitochondria. Moreover, apoptosis assays demonstrated that the overexpression of NsALD induces apoptosis in FHM cells. This study may lay the foundation for further exploration of the function of NsALD and facilitate further understanding of the pathogenic mechanism and the associated virulence factors of N. seriolae.

Peptide Bond Formation Between the Hetrosubunits of ω-Transaminase, Alanine Dehydrogenase, and Formate Dehydrogenase Through Subunit Splicing Promoted by Heterodimerization of Leucine Zipper Motifs.

For the multimeric enzymes R-ω-transaminase (RTA), alanine dehydrogenase (AlaDH), and formate dehydrogenase (FDH), peptide bond formation between the hetrosubunits has been achieved by the intein-mediated in vivo subunit splicing. The subunit ligation is triggered by the heterodimerization of an arginine rich leucine zipper motif with a glutamic acid rich leucine zipper motif. The one-by-one ligation of hetrosubunits constructs the pairing enzymes RTA&AlaDH and AlaDH&FDH. The ligation modes were analyzed based on blue native polyacrylamide gel electrophoresis (BN-PAGE). The spectra of circular dichroism (CD), fluorescence, and two-dimensional FTIR provide information on the secondary structures and stability of the pairing enzymes. The enzyme-substrate interaction was analyzed based on microscale thermophoresis analysis. In contrast to the mixed three enzymes RTA + AlaDH + FDH, the ligated enzymes RTA&AlaDH + AlaDH&FDH exhibited a much larger substrate affinity, higher stability, and significantly enhanced activity.

A novel type alanine dehydrogenase from Helicobacter aurati: Molecular characterization and application.

A novel alanine dehydrogenase (ADH; EC.1.4.1.1) with high pyruvate reduced activity was isolated from Helicobacter aurati and expressed in Escherichia coli BL21 (DE3). The optimum pH of the reduction and oxidation reaction were 8.0 and 9.0, respectively, and the optimum temperature was 55 °C. With pyruvate and alanine as substrates, the specific activity of HAADH1 were 268 U·mg-1 and 26 U·mg-1, respectively. HAADH1 had a prominent substrate specificity for alanine (Km = 2.23 mM, kcat/Km = 8.1 s-1·mM-1). In the reduction reaction, HAADH1 showed the highest substrate affinity for pyruvate (Km = 0.56 mM, kcat/Km = 364 s-1·mM-1). Compared to pyruvate, oxaloacetic acid, 2-ketobutyric acid, 3-fluoropyruvate, α-ketoglutaric acids, glyoxylic acid showed a residual activity of 93.30%, 8.93%, 5.62%, 2.57%, 2.51%, respectively. Phylogenetic tree analysis showed that this is a new type of ADH which have a low sequence similarity to available ADH reported in references. 3-Fluoropyruvate was effectively reduced to 3-fluoro-L-alanine by whole-cell catalysis.

LIOFeron®TB/LTBI: A novel and reliable test for LTBI and tuberculosis.

OBJECTIVES: High accuracy diagnostic screening tests for tuberculosis (TB) are required to improve the diagnosis of both active TB and latent Mycobacterium tuberculosis (MTB) infection (LTBI). The novel IGRA LIOFeron®TB/LTBI assay was tested and its accuracy was compared to the QuantiFERON®-TB Gold Plus assay. METHODS: A total of 389 subjects were enrolled in two cohorts and classified as healthy, active TB or LTBI persons. The blood of all the patients was tested with LIOFeron®TB/LTBI assay, containing MTB alanine dehydrogenase, able to differentiate active TB from LTBI diagnosis. The results obtained with both IGRAs, performed on the same 250 samples, were finally compared. RESULTS: The two assays demonstrated an excellent concordance of their results with patients' diagnosis of MTB infection. ROC analysis for QuantiFERON®-TB Gold Plus showed sensitivity and specificity respectively of 98% and 97% in diagnosing active TB patients and 85% and 94% in diagnosing LTBI subjects. LIOFeron®TB/LTBI assay showed sensitivity and specificity respectively of 90% and 98% in diagnosing active TB patients and 94% and 97% in diagnosing LTBI subjects. CONCLUSIONS: The two IGRAs displayed the same high accuracy in diagnosing MTB infection/TB disease, and LIOFeron®TB/LTBI assay demonstrated higher sensitivity than QuantiFERON®-TB Gold Plus test in LTBI detection.

Characterization of Alanine Dehydrogenase and Its Effect on Streptomyces coelicolorA3(2) Development in Liquid Culture.

Streptomyces, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of Streptomyces coelicolor A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lag-phase growth followed by a slow exponential growth of S. coelicolor due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from S. coelicolor was also heterologously cloned and expressed in Escherichia coli for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30°C, pH 9.5 with a catalytic efficiency, kcat/KM, of 2.0 ± 0.1 mM-1 s-1. The optimum enzyme activity for the reductive amination reaction was found at 30°C, pH 9.0 with a catalytic efficiency, kcat/KM, of 1.9 ± 0.1 mM-1 s-1.

Alanine dehydrogenases in mycobacteria.

Since NAD(H)-dependent L-alanine dehydrogenase (EC 1.1.4.1; Ald) was identified as one of the major antigens present in culture filtrates of Mycobacterium tuberculosis, many studies on the enzyme have been conducted. Ald catalyzes the reversible conversion of pyruvate to alanine with concomitant oxidation of NADH to NAD+ and has a homohexameric quaternary structure. Expression of the ald genes was observed to be strongly upregulated in M. tuberculosis and Mycobacterium smegmatis grown in the presence of alanine. Furthermore, expression of the ald genes in some mycobacteria was observed to increase under respiration-inhibitory conditions such as oxygen-limiting and nutrient-starvation conditions. Upregulation of ald expression by alanine or under respiration-inhibitory conditions is mediated by AldR, a member of the Lrp/AsnC family of transcriptional regulators. Mycobacterial Alds were demonstrated to be the enzymes required for utilization of alanine as a nitrogen source and to help mycobacteria survive under respiration-inhibitory conditions by maintaining cellular NADH/NAD+ homeostasis. Several inhibitors of Ald have been developed, and their application in combination with respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline was recently suggested.

Roles of Alanine Dehydrogenase and Induction of Its Gene in Mycobacterium smegmatis under Respiration-Inhibitory Conditions.

Here we demonstrated that the inhibition of electron flux through the respiratory electron transport chain (ETC) by either the disruption of the gene for the major terminal oxidase (aa3 cytochrome c oxidase) or treatment with KCN resulted in the induction of ald encoding alanine dehydrogenase in Mycobacterium smegmatis A decrease in functionality of the ETC shifts the redox state of the NADH/NAD+ pool toward a more reduced state, which in turn leads to an increase in cellular levels of alanine by Ald catalyzing the conversion of pyruvate to alanine with the concomitant oxidation of NADH to NAD+ The induction of ald expression under respiration-inhibitory conditions in M. smegmatis is mediated by the alanine-responsive AldR transcriptional regulator. The growth defect of M. smegmatis by respiration inhibition was exacerbated by inactivation of the ald gene, suggesting that Ald is beneficial to M. smegmatis in its adaptation and survival under respiration-inhibitory conditions by maintaining NADH/NAD+ homeostasis. The low susceptibility of M. smegmatis to bcc1 complex inhibitors appears to be, at least in part, attributable to the high expression level of the bd quinol oxidase in M. smegmatis when the bcc1-aa3 branch of the ETC is inactivated.IMPORTANCE We demonstrated that the functionality of the respiratory electron transport chain is inversely related to the expression level of the ald gene encoding alanine dehydrogenase in Mycobacterium smegmatis Furthermore, the importance of Ald in NADH/NAD+ homeostasis during the adaptation of M. smegmatis to severe respiration-inhibitory conditions was demonstrated in this study. On the basis of these results, we propose that combinatory regimens including both an Ald-specific inhibitor and respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline are likely to enable a more efficient therapy for tuberculosis.

Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties.

A link between carbon and nitrogen metabolism is important for serving as metabolic ancillary reactions. Here, we identified and characterized the alanine dehydrogenase gene in Aphanothece halophytica (ApalaDH) that is involved in alanine assimilation/dissimilation. Functional analysis revealed that ApalaDH encodes a bifunctional protein catalyzing the reversible reaction of pyruvate to L-alanine via its pyruvate reductive aminase (PvRA) activity, the reaction of L-alanine to pyruvate via its alanine oxidative dehydrogenase activity, and the non-reversible reaction of glyoxylate to glycine via its glyoxylate reductive aminase (GxRA) activity. Kinetic analysis showed the lowest affinity for pyruvate followed by L-alanine and glyoxylate with a Km of 0.22 ± 0.02, 0.72 ± 0.04, and 1.91 ± 0.43 mM, respectively. ApalaDH expression was upregulated by salt. Only PvRA and GxRA activities were detected in vivo and both activities increased about 1.2- and 2.7-fold upon salt stress. These features implicate that the assimilatory/dissimilatory roles of ApAlaDH are not only selective for L-alanine and pyruvate, but also, upon salt stress, can catabolize glyoxylate to generate glycine.

A conserved residue of l-alanine dehydrogenase from Bacillus pseudofirmus, Lys-73, participates in the catalytic reaction through hydrogen bonding.

A multiple protein sequence alignment of l-alanine dehydrogenases from different bacterial species revealed that five highly conserved amino acid residues Arg-15, Lys-73, Lys-75, His-96 and Asp-269 are potential catalytic residues of l-alanine dehydrogenase from Bacillus pseudofirmus OF4. In this study, recombinant OF4Ald and its mutants of five conserved residues were constructed, expressed in Escherichia coli, purified by His6-tag affinity column and gel filtration chromatography, structure homology modeling, and characterized. The purified protein OF4Ald displayed high specificity to l-alanine (15Umg-1) with an optimal temperature and pH of 40°C and 10.5, respectively. Enzymatic assay and activity staining in native gels showed that mutations at four conserved residue Arg-15, Lys-75, His-96 and Asp-269 (except residue Lys-73) resulted in a complete loss in enzymatic activity, which signified that these predicted active sites are indispensable for OF4Ald activity. In contrast, the mutant K73A resulted in 6-fold improvement in kcat/Km towards l-alanine as compared to the wild type protein. Further research of the residue Lys-73 substituted by various amino acids and structural modeling revealed that residue Lys-73 might be involved in the catalytic reaction of the enzyme by influencing the enzyme-substrate binding through the hydrogen-bonding interaction with conserved residue Lys-75.