IlvA1, a pyridoxal phosphate-dependent (PLP) enzyme, catalyzes the deamination of l-threonine and l-serine to yield 2-ketobutyric acid or pyruvate. To gain insights into the function of IlvA1, we determined its crystal structure from Pseudomonas aeruginosa to 2.3 Å. Density for a 2-ketobutyric acid product was identified in the active site and a putative allosteric site. Activity and substrate binding assays confirmed that IlvA1 utilizes l-threonine, l-serine, and L-allo-threonine as substrates. The enzymatic activity is regulated by the end products l-isoleucine and l-valine. Additionally, the efficiency of d-cycloserine and l-cycloserine inhibitors on IlvA1 enzymatic activity was examined. Notably, site-directed mutagenesis confirmed the active site residues and revealed that Gln165 enhances the enzyme activity, emphasizing its role in substrate access. This work provides crucial insights into the structure and mechanism of IlvA1 and serves as a starting point for further functional and mechanistic studies of the threonine deaminase in P. aeruginosa.

Threonine deaminase catalyzes the first step of isoleucine biosynthesis from threonine. In this protocol, we describe the process of measuring the enzymatic activity of threonine deaminase in the fission yeast cell lysate, which is catalyzed by Tda1. First, we describe the process of preparing cell lysates from fission yeast cell cultures. Subsequently, we explain how to measure the threonine deaminase activity using threonine or serine as a substrate. For complete details on the use and execution of this protocol, please refer to Sasaki et al. (2022).1.

Virus-like particles (VLPs) derived from bacteriophage P22 have been explored as biomimetic catalytic compartments. In vivo colocalization of enzymes within P22 VLPs uses sequential fusion to the scaffold protein, resulting in equimolar concentrations of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realizing the full potential of P22 VLPs as artificial metabolons. We present a tunable strategy for stoichiometric control over in vivo co-encapsulation of P22 cargo proteins, verified for fluorescent protein cargo by Förster resonance energy transfer. This was then applied to a two-enzyme reaction cascade. l-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesized from the readily available l-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We found that the loading density of both enzymes influences their activity, with higher activity found at lower loading density implying an impact of molecular crowding on enzyme activity. Conversely, increasing overall loading density by increasing the amount of threonine dehydratase can increase activity from the rate-limiting glutamate dehydrogenase. This work demonstrates the in vivo colocalization of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that controlled stoichiometry of individual enzymes in an enzymatic cascade is required for the optimal design of nanoscale biocatalytic compartments.

The hormones salicylic acid (SA) and jasmonic acid (JA) often act antagonistically in controlling plant defense pathways in response to hemibiotrophs/biotrophs (hemi/biotroph) and herbivores/necrotrophs, respectively. Threonine deaminase (TD) converts threonine to α-ketobutyrate and ammonia as the committed step in isoleucine (Ile) biosynthesis and contributes to JA responses by producing the Ile needed to make the bioactive JA-Ile conjugate. Tomato (Solanum lycopersicum) plants have two TD genes: TD1 and TD2. A defensive role for TD2 against herbivores has been characterized in relation to JA-Ile production. However, it remains unknown whether TD2 is also involved in host defense against bacterial hemi/biotrophic and necrotrophic pathogens. Here, we show that in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin flg22 peptide, an activator of SA-based defense responses, TD2 activity is compromised, possibly through carboxy-terminal cleavage. TD2 knockdown (KD) plants showed increased resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae but were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, suggesting TD2 plays opposite roles in response to hemibiotrophic and necrotrophic pathogens. This TD2 KD plant differential response to different pathogens is consistent with SA- and JA-regulated defense gene expression. flg22-treated TD2 KD plants showed high expression levels of SA-responsive genes, whereas TD2 KD plants treated with the fungal PAMP chitin showed low expression levels of JA-responsive genes. This study indicates TD2 acts negatively in defense against hemibiotrophs and positively against necrotrophs and provides insight into a new TD2 function in the elaborate crosstalk between SA and JA signaling induced by pathogen infection.

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2 to 3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.

Cysteine and homocysteine suppress the growth of Escherichia coli. It was explained by the inhibition of threonine deaminase (TD) (Harris in J Bacteriol 145(2):1031-1035, 1981). TD inhibition was detected by a decline in its product, 2-ketobutyrate (2-KB). We propose that cysteine or homocysteine may not inhibit TD activity. Instead, 2-KB binds to them forming stable cyclic adducts. This binding possibly leads to isoleucine limitation and growth inhibition.

Biomineralization is often used by microorganisms to sequester heavy metal ions and provides a potential means for remediating increasing levels of heavy metal pollution. Bacteria have been shown to utilize cysteine for the biomineralization of metal sulfide. Indeed, in the present study, the supplement of L-cysteine was found to significantly improve both cadmium resistance and removal abilities of a deep-sea bacterium Pseudomonas stutzeri 273 through cadmium sulfide (CdS) nanoparticle biomineralization. With a proteomic approach, threonine dehydratase of P. stutzeri 273 (psTD) was proposed to be a key factor enhancing bacterial cadmium resistance through catalyzing L-cysteine desulfuration, H2S generation and CdS nanoparticle biomineralization. Consistently, deletion of the gene encoding psTD in P. stutzeri 273 resulted in the decline of H2S generation, decrease of cadmium resistance, and reduction of cadmium removal ability, confirming the unique function of psTD directing the formation of CdS nanoparticles. Correspondingly, the single-enzyme biomineralization of CdS nanoparticle driven by psTD was further developed, and psTD was shown to act as a capping reagent for the mineralization reaction, which controlling the size and structure of nanocrystals. Our results provide important clues for the construction of engineered bacteria for cadmium bioremediation and widen the synthesis methods of nanomaterials.

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, D-lysine (D-Lys), in addition to the typical D-alanine (D-Ala) and D-glutamate (D-Glu). In a previous study, we identified a Lys racemase that is presumably associated with D-Lys biosynthesis. However, our understanding of D-amino acid metabolism in T. maritima and other bacteria remains limited, although D-amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5\'-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward L-threonine (L-Thr). It also acted on L-Ser and L-allo-Thr, but not on the corresponding D-amino acids. The catalytic mechanism did not follow typical Michaelis-Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency (kcat/K0.5) toward L-Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators L-valine and L-isoleucine (L-Ile) at low concentrations, while these L-amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of L-Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of L-Ile.