Medium-chain Alcohol Dehydrogenase Research Articles

Ancestor-originated engineering of medium-chain alcohol dehydrogenase enhances its catalytic compatibility by balancing activity, stability, and selectivity trade-offs

The role of medium-chain alcohol dehydrogenases (MDR) in the preparation of chiral drug intermediates is indispensable; however, limited activity and stability towards unnatural substrates are crucial factors that restrict their application. Further, research on the ancestral MDRs remains unclear. Therefore, enhancing the activity, selectivity, and stability of MDRs is necessary. In this study, we resurrected ancestors of the MDR family using ancestral sequence reconstruction with descendant GcADH as a probe. Among these ancestors, ancestor 136 exhibited increased thermal stability (ΔTm = 19.5 °C) and had a comparable conversion and selectivity as GcADH to the selected substrates. After in-silico screening using the Funclib tool, we obtained the mutant anc136-R2, which exhibited strengthened selectivity and activity towards N-Benzyl-3,3-difluoropiperidin-4-one (1a). Moreover, the mutations of anc136-R2 were simultaneously mapped to GcADH, resulting GcADH-R2. Based on the results of substrate spectrum characterisation, anc136 was more receptive to mutations. Specifically, for the substrates N-Boc-3-piperidone (4a) and N-boc-3-oxoazepane (7a), the conversion rates of anc136-R2 were 29 and 57 times higher, respectively, than those of GcADH-R2, without compromising selectivity. In conclusion, this work provides guidance for exploring the evolutionary trajectory of the MDR family and further illustrates the superior balanced “trade-off” of activity–selectivity–stability on ancestral enzymes.

Front Cover: Engineering a Medium‐chain Alcohol Dehydrogenase for Efficient Synthesis of (S)‐N−Boc‐3−pyrrolidinol by Adjusting the Conformational Dynamics of Loops (ChemCatChem 5/2023)

The Front Cover shows that a medium-chain alcohol dehydrogenase modified in the loop is efficiently catalyzing N-Boc-3-pyrrolidone to produce (S)-N-Boc-3-pyrrolidinol. In their Research Article, H. Wang and co-workers proposed that loop-loop interaction region acts like the “bridges” between loops and could be used to adjust conformational dynamics of the enzyme, thus modifying its catalytic efficiency. Combined with the analysis of residue orientation and interaction network, three loop-loop interaction regions were selected for mutagenesis. After screening, a mutant with 94.5-fold enhancement in catalytic efficiency was obtained. The results show that the residues located in the loop-loop interaction region could be used to effectively alter the loop conformation, thereby affecting the catalytic properties of the enzyme. More information can be found in the Research Article by H. Wang and co-workers.

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism.

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Using a novel data-driven combinatorial mutagenesis strategy to engineer an alcohol dehydrogenase for efficient geraniol synthesis

The monoterpene geraniol is widely applied in perfume, food, pharmaceutical, and other industries. However, conventional methods of geraniol production, including chemical synthesis and microbial fermentation, are associated with low selectivity and poor yields. Here, we engineered the medium-chain alcohol dehydrogenase AdhP from Escherichia coli K12 as a biocatalyst for the efficient bioconversion of geranial into geraniol. First, a single point mutation library targeting 12 key residues identified based on computational approaches was constructed, and 24 positive mutants with improved reducing activity toward geranial were obtained. Subsequently, we established a data-driven combinatorial mutagenesis strategy for five rounds of engineering with reference to a mathematical “weighting” principle, yielding the six-point mutant M6, which exhibited 8.5-fold higher catalytic efficiency than the wild type. In silico analysis revealed its enhanced structural stability and shortened critical catalytic distances. Recombinant bacterias co-expressing M6 and glucose dehydrogenase were employed as biocatalysts for the reduction of geranial. As a result, 100.0 g/L of geranial was converted to geraniol in 8 h with a space-time yield of up to 291.3 g·L−1d−1. This study provides a multipoint mutant library construction strategy for the accumulation of beneficial mutations to tailor enzymes and provides a potential biocatalyst for efficient geraniol synthesis.

Genome-wide identification of alcohol dehydrogenase (ADH) gene family under waterlogging stress in wheat (Triticum aestivum).

BackgroundAlcohol dehydrogenase (ADH) plays an important role in plant survival under anaerobic conditions. Although some research about ADH in many plants have been carried out, the bioinformatics analysis of the ADH gene family from Triticum aestivum and their response to abiotic stress is unclear.MethodsA total of 22 ADH genes were identified from the wheat genome, and these genes could be divided into two subfamilies (subfamily I and subfamily II). All TaADH genes belonged to the Medium-chain ADH subfamily. Sequence alignment analysis showed that all TaADH proteins contained a conservative GroES-like domain and Zinc-binding domain. A total of 64 duplicated gene pairs were found, and the Ka/Ks value of these gene pairs was less than 1, which indicated that these genes were relatively conservative and did not change greatly in the process of duplication.ResultsThe organizational analysis showed that nine TaADH genes were highly expressed in all organs, and the rest of TaADH genes had tissue specificity. Cis-acting element analysis showed that almost all of the TaADH genes contained an anaerobic response element. The expression levels of ADH gene in waterlogging tolerant and waterlogging sensitive wheat seeds were analyzed by quantitative real-time PCR (qRT-PCR). This showed that some key ADH genes were significantly responsive to waterlogging stress at the seed germination stage, and the response of waterlogging tolerant and waterlogging sensitive wheat seeds to waterlogging stress was regulated by different ADH genes. The results may be helpful to further study the function of TaADH genes and to determine the candidate gene for wheat stress resistance breeding.

Early and Late Steps of Quinine Biosynthesis.

The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3. Additionally, the discovery of an O-methyltransferase specific for 6′-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.

Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives.

The zinc‐dependent medium‐chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2‐oxo‐4‐phenylbutyrate) opens up new routes in the synthesis of ethyl‐2‐hydroxy‐4‐phenylbutanoate, an important intermediate for anti‐hypertension drugs like enalaprilat and lisinopril. We have performed computer‐aided engineering of ReADH toward ethyl 2‐oxo‐4‐phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2‐oxo‐4‐phenylbutyrate, octan‐3‐one or octan‐4‐one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site‐saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6‐fold improved activity toward ethyl 2‐oxo‐4‐phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16‐fold increased activity toward octan‐4‐one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30‐fold decreased activity toward octan‐2‐one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

Green and scalable synthesis of chiral aromatic alcohols through an efficient biocatalytic system.

SummaryChiral aromatic alcohols have received much attention due to their widespread use in pharmaceutical industries. In the asymmetric synthesis processes, the excellent performance of alcohol dehydrogenase makes it a good choice for biocatalysts. In this study, a novel and robust medium‐chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyse the asymmetric reduction of aromatic ketones to chiral aromatic alcohols. The reduction of 2‐hydroxyacetophenone (2‐HAP) to (R)‐(‐)‐1‐phenyl‐1,2‐ethanediol ((R)‐PED) was chosen as a template to evaluate its catalytic activity. A specific activity of 110 U mg−1 and a 99% purity of e.e. was achieved in the presence of NADH. An efficient bienzyme‐coupled catalytic system (RhADH and formate dehydrogenase, CpFDH) was established using a two‐phase strategy (dibutyl phthalate and buffer), which highly raised the tolerated substrate concentration (60 g l−1). Besides, a broad range of aromatic ketones were enantioselectively reduced to the corresponding chiral alcohols by this enzyme system with highly enantioselectivity. This system is of the potential to be applied at a commercial scale.

The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns.

Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and –insensitive groups, and the functions of CmADHs were discussed.

The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress.

Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin.

Oxidation of ethylene glycol to glycolaldehyde using a highly selective alcohol dehydrogenase from Gluconobacter oxydans

A cytosolic medium-chain alcohol dehydrogenase Gox0313 from Gluconobacter oxydans DSM2003 was heterologously expressed in Escherichia coli BL21 (DE3) and the resulting proteins were purified and characterized. The recombinant enzyme was confirmed to have a good ability to selectively oxidize the terminal hydroxyl group of varied aliphatic and aromatic diols to the corresponding hydroxyl aldehydes, with no oxidative activities toward hydroxyl aldehydes in the presence of NAD+. This enzyme could not oxidize sec-alcohols. Among the primary diols, ethylene glycol was efficiently converted to glycolaldehyde by Gox0313 coupled with NADH oxidase-2 (NOX-2) from Lactobacillus brevis ATCC367 for NAD+ regeneration. Under the optimal conditions using this enzyme-coupled system (1U/mL Gox0313 and 4U/mL NOX-2), 42.32mM glycolaldehyde was produced from 50mM ethylene glycol in 8h with a yield of 83.2%. When the enzyme concentration of Gox0313 was increased to 120U/mL in the reaction, 500mM of ethylene glycol was converted to 484.2mM of glycolaldehyde in 14h, resulting in a yield of 96.8%. With respect to the superior catalytic activity to primary diols, Gox0313 might be a potentially promising biocatalyst for bioproduction of glycolaldehyde and other hydroxyl aldehydes.

Unconserved substrate-binding sites direct the stereoselectivity of medium-chain alcohol dehydrogenase

Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs' stereoselectivity.

Expression, purification, and characterization of formaldehyde dehydrogenase from Pseudomonas aeruginosa

As a member of zinc-containing medium-chain alcohol dehydrogenase family, formaldehyde dehydrogenase (FDH) can oxidize toxic formaldehyde to less active formate with NAD+ as a cofactor and exists in both prokaryotes and eukaryotes. Most FDHs are well known to be glutathione-dependent in the catalysis of formaldehyde oxidation, but the enzyme from Pseudomonas putida is an exception, which is independent of glutathione. To identify novel glutathione-independent FDHs from other bacterial strains and facilitate the corresponding structural and enzymatic studies, high-level soluble expression and efficient purification of these enzymes need to be achieved. Here, we present molecular cloning, expression, and purification of the FDH from Pseudomonas aeruginosa, which is a Gram-negative pathogenic bacterium causing opportunistic human infection. The FDH of P. aeruginosa shows high sequence identity (87.97%) with that of P. putida. Our results indicated that coexpression with molecular chaperones GroES, GroEL, and Tig has significantly attenuated inclusion body formation and improved the solubility of the recombinant FDH in Escherichiacoli cells. A purification protocol including three chromatographic steps was also established to isolate the recombinant FDH to homogeneity with a yield of ∼3.2mg from 1L of cell culture. The recombinant P. aeruginosa FDH was properly folded and biologically functional, as demonstrated by the mass spectrometric, crystallographic, and enzymatic characterizations of the purified proteins.

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6.

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=88.35, b=128.73, c=131.03 Å.

Structure of formaldehyde dehydrogenase from Pseudomonas aeruginosa: the binary complex with the cofactor NAD+.

Formaldehyde dehydrogenase (FDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family which oxidizes toxic formaldehyde to formate using NAD(+) as an electron carrier. Three-dimensional structures have been reported for FDHs from several different species. Most FDHs are dependent on glutathione for catalysis, but the enzyme from Pseudomonas putida is an exception. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.7 Å resolution of FDH from P. aeruginosa are described. Both the tetrameric assembly and the NAD(+)-binding mode of P. aeruginosa FDH are similar to those of P. putida FDH, which is in good agreement with the high sequence identity (87.97%) between these two proteins. Preliminary enzymatic kinetics studies of P.aeruginosa FDH also revealed a conserved glutathione-independent `ping-pong' mechanism of formaldehyde oxidization.

Origin and evolution of medium chain alcohol dehydrogenases

Different lines of alcohol dehydrogenases (ADHs) have separate superfamily origins, already recognized but now extended and re-evaluated by re-screening of the latest databank update. The short-chain form (SDR) is still the superfamily with most abundant occurrence, most multiple divergence, most prokaryotic emphasis, and most non-complicated architecture. This pattern is compatible with an early appearance at the time of the emergence of prokaryotic cellular life. The medium-chain form (MDR) is also old but second in terms of all the parameters above, and therefore compatible with a second emergence. However, this step appears seemingly earlier than previously considered, and may indicate sub-stages of early emergences at the increased resolution available from the now greater number of data entries. The Zn-MDR origin constitutes a third stage, possibly compatible with the transition to oxidative conditions on earth. Within all these three lines, repeated enzymogeneses gave the present divergence. MDR–ADH origin(s), at a fourth stage, may also be further resolved in multiple or extended modes, but the classical liver MDR–ADH of the liver type can still be traced to a gene duplication ∼550MYA (million years ago), at the early vertebrate radiation, compatible with the post-eon-shift, “Cambrian explosion”. Classes and isozymes correspond to subsequent and recent duplicatory events, respectively. They illustrate a peculiar pattern with functional and emerging evolutionary distinctions between parent and emerging lines, suggesting a parallelism between duplicatory and mutational events, now also visible at separate sub-stages. Combined, all forms show distinctive patterns at different levels and illustrate correlations with global events. They further show that simple molecular observations on patterns, multiplicities and occurrence give much information, suggesting common divergence rules not much disturbed by horizontal gene transfers after the initial origins.

Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases

Two genes encoding recombinant cytosolic oxidoreductases from Gluconobacter oxydans, gox0313 and gox0646, were heterologously expressed in Escherichia coli and the resulting proteins were purified and characterized. GOX0313 was identified as a medium-chain alcohol dehydrogenase, whereas GOX0646 was classified as a ketocarbonyl reductase. GOX0313 had a broad substrate spectrum and oxidized various primary alcohols. However, GOX0313 had a preference for substrate reduction, reducing many aldehydes and α-diketones. In contrast, GOX0646 had a narrow substrate spectrum and reduced α-diketones, preferring short-chain ketocarbonyls. Both enzymes regio- and stereospecifically reduced α-diketones to the corresponding (S)-hydroxy ketone, as shown by NMR. These products are difficult to produce chemically, requiring complicated protecting group chemistry. Furthermore, hydroxy ketones find industrial application in the production of pheromones, fragrances, flavors, and pharmaceuticals. Hence, these enzymes are interesting biocatalysts for the production of enantiomerically pure building blocks that are difficult to prepare chemically.

Production of a thermostable alcohol dehydrogenase from Rhodococcus ruber in three different yeast species using the Xplor®2 transformation/expression platform

The Xplor®2 transformation/expression platform was employed for comparative assessment of three different yeast species as hosts for synthesis of a thermostable nicotinamide adenine dinucleotide (NAD⁺)-dependent medium-chain alcohol dehydrogenase from Rhodococcus ruber strain 219. Using yeast ribosomal DNA (rDNA) integrative expression cassettes (YRCs) and yeast integrative expression cassettes (YICs) equipped with a selection-marker module and one, two or four expression modules for transformation of auxotrophic Arxula adeninivorans, Hansenula polymorpha, and Saccharomyces cerevisiae strains, quantitative comparison of the yield of recombinant alcohol dehydrogenase RR-ADH6Hp in all three species was carried out. In all cases, the RR-ADH6H gene was expressed under the control of the strong constitutive A. adeninivorans-derived TEF1 promoter, which functions in all yeast species analyzed. Recombinant RR-ADH6Hp accumulated intracellularly in all strains tested. The best yields of active enzyme were obtained from A. adeninivorans, with S. cerevisiae producing intermediate amounts. Although H. polymorpha was the least efficient producer overall, the product obtained was most similar to the enzyme synthesized by R. ruber 219 with respect to its thermostability.

Characterization of new medium-chain alcohol dehydrogenases adds resolution to duplications of the class I/III and the sub-class I genes

Four additional variants of alcohol and aldehyde dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR alcohol dehydrogenases from characterizations of the pigeon ( Columba livia) and dogfish ( Scyliorhinus canicula) major liver alcohol dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.

Highly selective l-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of l-threonine

l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD +-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and d l-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD +-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.