Efficient synthesis of L-malic acid by malic enzyme biocatalysis with CO2 fixation.

The malic enzyme (ME) catalyzes the synthesis of L-malic acid (L-MA) from pyruvic acid and CO2 with NADH as the reverse reaction of L-MA decarboxylation. Carboxylation requires excess pyruvic acid, limiting its application. In this study, it was determined that CO2 was the carboxyl donor by parsing the effects of HCO3- and CO2, which provided a basis for improving the L-MA yield. Moreover, the concentration ratio of pyruvic acid to NADH was reduced from 70:1 to 5:1 using CO2 to inhibit decarboxylation and to introduce the ME mutant A464S with a 2-fold lower Km than that of the wild type. Finally, carboxylation was coupled with NADH regeneration, resulting in a maximum L-MA yield of 77 % based on the initial concentration of pyruvic acid. Strategic modifications, including optimal reactant ratios and efficient mutant ME, significantly enhanced L-MA synthesis from CO2, providing a promising approach to the biotransformation process.

A Dual-Cascade Activatable Near-Infrared Fluorescent Probe for Precise Intraoperative Imaging of Tumor.

Accurate intraoperative tumor delineation is critical to achieving successful surgical outcomes. However, conventional techniques typically suffer from poor specificity and low sensitivity and are time-consuming, which greatly affects intraoperative decision-making. Here, we report a cascade activatable near-infrared fluorescent (NIRF) probe IR780SS@CaP that can sequentially respond to tumor acidity and elevated glutathione levels for accurate intraoperative tumor localization. Compared with nonactivatable and single-factor activatable probes, IR780SS@CaP with a cascade strategy can minimize nonspecific activation and false positive signals in a complicated biological environment, affording a superior tumor-to-normal tissue ratio to facilitate the delineation of abdominal metastases. Small metastatic lesions that were less than 1 mm in diameter can be precisely identified by IR780SS@CaP and completely excised under NIRF imaging guidance. This study could benefit tumor diagnosis and image-guided tumor surgery by providing real-time information and reliable decision support, thus reducing the risk of both recurrence and complications to improve patient outcomes.

An Activatable Near-Infrared Fluorescent Probe for Precise Detection of the Pulmonary Metastatic Tumors: A Traditional Molecule Having a Stunning Turn.

An accurate detection of lung metastasis is of great significance for making better treatment choices and improving cancer prognosis, but remains a big challenge in clinical practice. In this study, we propose a reinventing strategy to develop a pH-activatable near-infrared (NIR) fluorescent nanoprobe, pulmonary metastasis tracer (denoted as PMT), based on assembly of NIR dye IR780 and calcium phosphate (CaP). By delicately tuning the intermolecular interactions during the assembly process and dye doping content, as well as the synthetic condition of probe, the fluorescence of PMT could be finely adjusted via the tumor acidity-triggered disassembly. Notably, the selected PMT9 could sharply convert subtle pH variations into a distinct fluorescence signal to generate high fluorescence ON/OFF contrast, dramatically reducing the background signals. Benefiting from such preferable features, PMT9 is able to precisely identify not only the tumor sites in orthotopic lung cancer models but also the pulmonary metastases in mice with remarkable signal-to-background ratio (SBR). This study provides a unique strategy to turn shortcomings of traditional dye IR780 during in vivo imaging into advantages and further expand the application of fluorescent probe to image lung associated tumor lesions.

The catalytic mechanism of direction-dependent interactions for 2,3-dihydroxybenzoate decarboxylase.

Benzoic acid decarboxylases offer an elegant alternative to CO2 fixation by reverse reaction-carboxylation, which is named the bio-Kolbe-Schmitt reaction, but they are unfavorable to carboxylation. Enhancing the carboxylation efficiency of reversible benzoic acid decarboxylases is restricted by the unexplained carboxylation mechanisms. The direction of reversible enzyme catalytic reactions depends on whether catalytic residues at the active center of the enzyme are protonated, which is subjected by the pH. Therefore, the forward and reverse reactions could be separated at different pH values. Reversible 2,3-dihydroxybenzoate acid decarboxylase undergoes decarboxylation at pH 5.0 and carboxylation at pH 8.6. However, it is unknown whether the interaction of enzymes with substrates and products in the forward and reverse reactions can be exploited to improve the catalytic activity of reversible enzymes in the unfavorable direction. Here, we identify a V-shaped tunnel of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) through which the substrate travels in the enzyme, and demonstrate that the side chain conformation of a tyrosine residue controls the entry and exit of substrate/product during reversible reactions. Together with the kinetic studies of the mutants, it is clarified that interactions between substrate/product traveling through the enzyme tunnel in 2,3-DHBD_Ao are direction-dependent. These results enrich the understanding of the interactions of substrates/products with macromolecular reversible enzymes in different reaction directions, thereby demonstrating a possible path for engineering decarboxylases with higher carboxylation efficiency. KEY POINTS: • The residue Trp23 of 2,3-DHBD_Ao served as a switch to control the entry and exit of catechol • A V-shaped tunnel of 2,3-DHBD_Ao for decarboxylation and carboxylation reactions was identified • The results provide a promising strategy for engineering decarboxylases with direction-dependent residues inside the substrate/product traveling tunnel of the enzyme.

Unique recognition of the microalgal plastidial glycerol-3-phosphate acyltransferase for acyl-ACP.

Plastidial glycerol-3-phosphate acyltransferases (GPATs) catalyze acyl-ACP and glycerol-3-phosphate to synthesize lysophosphatidic acid in vivo, which initiates the formation of various glycerolipids. Although the physiological substrates of plastidial GPATs are acyl-ACPs, acyl-CoAs have been commonly studied on the GPATs in vitro. However, little is known whether there are any distinct features of GPATs towards acyl-ACP and acyl-CoA. In this study, the results showed that the microalgal plastidial GPATs preferred acyl-ACP to acyl-CoA, while surprisingly, the plant-derived plastidial GPATs showed no obvious preferences towards these two acyl carriers. The key residues responsible for the distinct feature of microalgal plastidial GPATs were compared with plant-derived plastidial GPATs in their efficiency to catalyze acyl-ACP and acyl-CoA. Microalgal plastidial GPATs uniquely recognized acyl-ACP as compared to with other acyltransferases. The structure of the acyltransferases-ACP complex highlights only the involvement of the large structural domain in ACP in microalgal plastidial GPAT while in the other acyltransferases, both large and small structural domains were involved in the recognition process. The interaction sites on the plastidial GPAT from the green alga Myrmecia incisa (MiGPAT1) with ACP turned out to be K204, R212 and R266. A unique recognition between the microalgal plastidial GPAT and ACP was elucidated.

Conformational Changes of Acyl Carrier Protein Switch the Chain Length Preference of Acyl-ACP Thioesterase ChFatB2.

Microbial fatty acids are synthesized by Type II fatty acid synthase and could be tailored by acyl-ACP thioesterase. With the prospects of medium-chain fatty-acid-derivative biofuels, the selectivity of thioesterase has been studied to control the fatty acid product chain length. Here, we report an alternative approach by manipulating the acyl carrier protein portion of acyl-ACP to switch the chain length propensity of the thioesterase. It was demonstrated that ChFatB2 from Cuphea hookeriana preferred C10-ACP to C8-ACP with ACP from E. coli, while converting preference to C8-ACP with ACP from Cuphea lanceolate. Circular dichroism (CD) results indicated that the C8-EcACP encountered a 34.4% α-helix increment compared to C10-EcACP, which resulted in an approximate binding affinity decrease in ChFatB2 compared to C10-EcACP. Similarly, the C10-ClACP2 suffered a 45% decrease in helix content compared to C8-ClACP2, and the conformational changes resulted in an 18% binding affinity decline with ChFatB2 compared with C10-ClACP2. In brief, the study demonstrates that the ACP portion of acyl-ACP contributes to the selectivity of acyl-ACP thioesterase, and the conformational changes of EcACP and ClACP2 switch the chain length preference of ChFatB2 between C8 and C10. The result provides fundamentals for the directed synthesis of medium-chain fatty acids based on regulating the conformational changes of ACPs.

Co-Expression of Lipid Transporters Simultaneously Enhances Oil and Starch Accumulation in the Green Microalga Chlamydomonas reinhardtii under Nitrogen Starvation.

Lipid transporters synergistically contribute to oil accumulation under normal conditions in microalgae; however, their effects on lipid metabolism under stress conditions are unknown. Here, we examined the effect of the co-expression of lipid transporters, fatty acid transporters, (FAX1 and FAX2) and ABC transporter (ABCA2) on lipid metabolism and physiological changes in the green microalga Chlamydomonas under nitrogen (N) starvation. The results showed that the TAG content in FAX1-FAX2-ABCA2 over-expressor (OE) was 2.4-fold greater than in the parental line. Notably, in FAX1-FAX2-ABCA2-OE, the major membrane lipids and the starch and cellular biomass content also significantly increased compared with the control lines. Moreover, the expression levels of genes directly involved in TAG, fatty acid, and starch biosynthesis were upregulated. FAX1-FAX2-ABCA2-OE showed altered photosynthesis activity and increased ROS levels during nitrogen (N) deprivation. Our results indicated that FAX1-FAX2-ABCA2 overexpression not only enhanced cellular lipids but also improved starch and biomass contents under N starvation through modulation of lipid and starch metabolism and changes in photosynthesis activity. The strategy developed here could also be applied to other microalgae to produce FA-derived energy-rich and value-added compounds.

A metabolic acidity-activatable calcium phosphate probe with fluorescence signal amplification capabilities for non-invasive imaging of tumor malignancy.

Dysregulated energy metabolism has recently been recognized as an emerging hallmark of cancer. Tumor cells, which are characterized by abnormal glycolysis, exhibit a lower extracellular pH (6.5-7.0) than normal tissues (7.2-7.4), providing a promising target for tumor-specific imaging and therapy. However, most pH-sensitive materials are unable to distinguish such a subtle pH difference owing to their wide and continuous pH-responsive range. In this study, we developed an efficient strategy for the fabrication of a tumor metabolic acidity-activatable calcium phosphate (CaP) fluorescent probe (termed MACaP9). Unlike traditional CaP-based biomedical nanomaterials, which only work within more acidic organelles, such as endosomes and lysosomes (pH 4.0-6.0), MACaP9 could not only specifically respond to the tumor extra-cellular pH but also rapidly convert pH variations into a distinct fluorescence signal to visually distinguish tumor from normal tissues. The superior sensitivity and specificity of MACaP9 enabled high-contrast visualization of a broad range of tumors, as well as small tumor lesions.

Estimating dynamic vascular perfusion based on Er-based lanthanide nanoprobes with enhanced down-conversion emission beyond 1500 nm.

Peripheral artery disease (PAD) is a common, yet serious, circulatory condition that can increase the risk of amputation, heart attack or stroke. Accurate identification of PAD and dynamic monitoring of the treatment efficacy of PAD in real time are crucial for optimizing therapeutic outcomes. However, current imaging techniques do not enable these requirements. Methods: A lanthanide-based nanoprobe with emission in the second near-infrared window b (NIR-IIb, 1500-1700 nm), Er-DCNPs, was utilized for continuous imaging of dynamic vascular structures and hemodynamic alterations in real time using PAD-related mouse models. The NIR-IIb imaging capability, stability, and biocompatibility of Er-DCNPs were evaluated in vitro and in vivo. Results: Owing to their high temporal-spatial resolution in the NIR-IIb imaging window, Er-DCNPs not only exhibited superior capability in visualizing anatomical and pathophysiological features of the vasculature of mice but also provided dynamic information on blood perfusion for quantitative assessment of blood recovery, thereby achieving the synergistic integration of diagnostic and therapeutic imaging functions, which is very meaningful for the successful management of PAD. Conclusion: Our findings indicate that Er-DCNPs can serve as a promising system to facilitate the diagnosis and treatment of PAD as well as other vasculature-related diseases.

Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide.

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.

Manipulating fatty-acid profile at unit chain-length resolution in the model industrial oleaginous microalgae Nannochloropsis.

The chain length (CL) of fatty acids (FAs) is pivotal to oil property, yet to what extent it can be customized in industrial oleaginous microalgae is unknown. In Nannochloropsis oceanica, to modulate long-chain FAs (LCFAs), we first discovered a fungi/bacteria-originated polyketide synthase (PKS) system which involves a cytoplasmic acyl-ACP thioesterase (NoTE1). NoTE1 hydrolyzes C16:0-, C16:1- and C18:1-ACP in vitro and thus intercepts the specific acyl-ACPs elongated by PKS for polyunsaturated FA biosynthesis, resulting in elevation of C16/C18 monounsaturated FAs when overproduced and increase of C20 when knocked out. For medium-chain FAs (MCFAs; C8-C14), C8:0 and C10:0 FAs are boosted by introducing a Cuphea palustris acyl-ACP TE (CpTE), whereas C12:0 elevated by rationally engineering CpTE enzyme's substrate-binding pocket to shift its CL preference towards C12:0. A mechanistic model exploiting both native and engineered PKS and type II FAS pathways was thus proposed for manipulation of carbon distribution among FAs of various CL. The ability to tailor FA profile at the unit CL resolution from C8 to C20 in Nannochloropsis spp. lays the foundation for scalable production of designer lipids via industrial oleaginous microalgae.

Characterization of the substrate scope of an alcohol dehydrogenase commonly used as methanol dehydrogenase.

Many alcohol dehydrogenases (ADHs) catalyze oxidation of a broad scope of alcohols. When an NAD-dependent ADH oxidizes methanol, albeit at a poor rate, it may be treated as methanol dehydrogenase (MDH). One ADH from Geobacillus stearothermophilus DSM 2334 (GsADH) has been widely used as MDH, but its actual substrate scope remains less characterized. Here we purified recombinant GsADH from Escherichia coli and determined its crystal structure. We collected kinetics data of this enzyme towards a number of short chain alcohols, and found that isopropanol is by far the most favorable substrate. Moreover, molecular docking analysis suggested that substrate preference is mainly attributed to the conformer energy of the protein-substrate complex. Our data clarified the substrate scope of GsADH and provided structural insights, which may facilitate more efficient cofactor regeneration and rational metabolic engineering.

Structure and catalytic mechanistic insight into Enterobacter aerogenes acetolactate decarboxylase.

α-Acetolactate decarboxylase (ALDC) catalyses α-acetolactate into acetoin (3-hydroxy-2-butanone, AC) and is considered to be the rate-limiting enzyme in the synthesis of 2,3-butanediol. In this work, the enzymatic activity of ALDC from Enterobacter aerogenes ALDC (E.a.-ALDC) was fully characterized with enzyme kinetics, indicating a Km of 14.83 ± 0.87 mM and a kcat of 0.81 ± 0.09 s-1. However, compared with the activities of ALDCs reported from other bacteria, the activity of E.a.-ALDC was determined to present a relatively lower value of 849.08 ± 35.21 U/mg. The enzyme showed maximum activity at pH 5.5. In addition, the activity of E.a.-ALDC was promoted by Mg2+. The crystal structure of E.a.-ALDC firstly solved by X-ray crystallography at resolution of 2.4 Å revealed a chelated zinc ion with conserved His199, His201, His212, Glu70 and Glu259. In the active center, the conservative Arg150 was particularly proven to deviate from the zinc ion of the active centre, by adopting a flexible conformational change, resulting in a weak interaction network of the enzyme and the substrate. Further in silico docking of E.a.-ALDC with two enantiomers, (R)-acetolactate and (S)-acetolactate, unaltered the interaction network of E.a.-ALDC from the apo structure, which confirmed the weakened role of Arg150 in the catalytic properties of E.a.-ALDC. Our results reveal a unique structure-function relationship of acetolactate decarboxylase and provide a fundamental basis for the enzymatic synthesis of acetoin.

Expanding of Phospholipid:Diacylglycerol AcylTransferase (PDAT) from Saccharomyces cerevisiae as Multifunctional Biocatalyst with Broad Acyl Donor/Acceptor Selectivity.

Triacylglycerols are considered one of the most promising feedstocks for biofuels. Phospholipid:diacylglycerol acyltransferase (PDAT), responsible for the last step of triacylglycerol synthesis in the acyl-CoA-independent pathway, has attracted much attention by catalyzing membrane lipid transformation. However, due to lack of biochemical and enzymatic studies, PDAT has not carried forward in biocatalyst application. Here, the PDAT from Saccharomyces cerevisiae was expressed in Pichia pastoris. The purified enzymes were studied using different acyl donors and acceptors by thin layer chromatography and gas chromatography. In addition of the preferred acyl donor of PE and PC, the results identified that ScPDAT was capable of using broad acyl donors such as PA, PS, PG, MGDG, DGDG, and acyl-CoA, and ScPDAT was more likely to use unsaturated acyl donors comparing 18:0/18:1 to 18:0/18:0 phospholipids. With regard to acyl acceptors, ScPDAT preferred 1,2 to 1,3-diacylglycerol (DAG), while 12:0/12:0 DAG was identified as the optimal acyl acceptor, followed by 18:1/18:1 and 18:1/16:0 DAG. Additionally, ScPDAT reveals esterification activity that can utilize methanol as acyl acceptor to generate fatty acid methyl esters. The results fully expand the enzymatic selectivity of ScPDAT and provide fundamental knowledge for synthesis of triacylglycerol-derived biofuels.

Stereoselective catalysis controlled by a native leucine or variant isoleucine wing-gatekeeper in 2-haloacid dehalogenase.

Comprehensively understanding enzymatic stereoselectivity will assist in the creation of new enzymes for producing optically pure compounds for chemical applications. The essential features for selecting enantiomers are controlled by particular residues or regions of the enzymes. We report a stereoselective mechanism in the D-2-haloacid dehalogenase HadD AJ1, in which L288 is identified as a gatekeeper in the access channel that strictly recognizes D-enantiomers. Mutagenesis of L288 to isoleucine (I) enlarges the size of the channel and allows the enzyme to accommodate L-enantiomers. Furthermore, the wing flip of I288 induces hydrophobic interactions with the L-enantiomer and directly affects the catalytic efficiency. The results illustrate the dynamic catalytic mechanisms of Leu-Ile gatekeepers and provide knowledge for unveiling the basis of stereospecificity in biocatalysts.

SGIP1 dimerizes via intermolecular disulfide bond in µHD domain during cellular endocytosis.

Along with its homologs FCHo1 and FCHo2, SGIP1 plays an important role in clathrin-mediated endocytosis. The highly conserved C-terminal µHD domains in these proteins are the critical regions interacting with adapter molecules such as Eps15. The crystal structure of µHD domain of SGIP1 has been reported previously. In this study, we found that µHD domain of SGIP1 is capable of forming a stable dimer by an intermolecular disulfide bond formed by C632 in our crystal structure. The mutational study of C632 revealed that this residue is important for the function of SGIP1 during cellular endocytosis. Our study revealed a new dimerization and/or oligomerization manner in theses adaptor proteins, which is a critical prerequisite for their proper function.

Structural and enzymatic characterization of acetolactate decarboxylase from Bacillus subtilis.

Acetoin is an important physiological metabolite excreted by microbes. Its functions include avoiding acidification, participating in regulation of the NAD+/NADH ratio, and storing carbon. Acetolactate decarboxylase is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin). It catalyzes conversion of the (R)- and (S)-enantiomers of acetolactate to generate the single product, (R)-acetoin. In addition to the X-ray crystal structure of acetolactate decarboxylase from Bacillus brevis, although the enzyme is widely present in microorganisms, very few atomic structures of acetolactate decarboxylase are reported. In this paper, we solved and reported a 1.5 Å resolution crystal structure of acetolactate decarboxylase from Bacillus subtilis. Dimeric assembly is observed in the solved structure, which is consistent with the elution profile conducted by molecular filtration. A zinc ion is coordinated by highly conserved histidines (191, 193, and 204) and conserved glutamates (62 and 251). We performed kinetic studies on acetolactate decarboxylase from Bacillus subtilis using circular dichroism, allowing the conversion of acetolactate to chiral acetoin for real-time tracking, yielding a Km value of 21 mM and a kcat value of 2.2 s-1. Using the two enantiomers of acetolactate as substrates, we further investigated the substrate preference of acetolactate decarboxylase from Bacillus subtilis by means of molecular docking and dynamic simulation in silico. The binding free energy of (S)-acetolactate was found to be ~ 30 kcal/mol greater than that of (R)-acetolactate, indicating a more stable binding for (S)-acetolactate.

Tuning of acyl-ACP thioesterase activity directed for tailored fatty acid synthesis.

Medium-chain fatty acids have attracted significant attention as sources of biofuels in recent years. Acyl-ACP thioesterase, which is considered as the key enzyme to determine the carbon chain length, catalyzes the termination of de novo fatty acid synthesis. Although recombinant medium-chain acyl-ACP thioesterase (TE) affects the fatty acid profile in heterologous cells, tailoring of the fatty acid composition merely by engineering a specific TE is still intractable. In this study, the activity of a C8-C10-specific thioesterase FatB2 from Cuphea hookeriana on C10-ACP was quantified twice as high as that on C8-ACP based on a synthetic C8-C16 acyl-ACP pool in vitro. Whereas in vivo, it was demonstrated that ChFatB2 preferred to accumulate C8 fatty acids with 84.9% composition in the ChFatB2-engineered E. coli strain. To achieve C10 fatty acid production, ChFatB2 was rationally tuned based on structural investigation and enzymatic analysis. An I198E mutant was identified to redistribute the C8-ACP flow, resulting in C10 fatty acid being produced as the principal component at 57.6% of total fatty acids in vivo. It was demonstrated that the activity of TE relative to ß-ketoacyl-ACP synthases (KAS) directly determined the fatty acid composition. Our results provide a prospective strategy in tailoring fatty acid synthesis by tuning of TE activities based on TE-ACP interaction.

Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures.

D-2-haloacid dehalogenases (D-DEXs) catalyse the hydrolytic dehalogenation of D-2-haloacids, releasing halide ions and producing the corresponding 2-hydroxyacids. A structure-guided elucidation of the catalytic mechanism of this dehalogenation reaction has not been reported yet. Here, we report the catalytic mechanism of a D-DEX, HadD AJ1 from Pseudomonas putida AJ1/23, which was elucidated by X-ray crystallographic analysis and the H218O incorporation experiment. HadD AJ1 is an α-helical hydrolase that forms a homotetramer with its monomer including two structurally axisymmetric repeats. The product-bound complex structure was trapped with L-lactic acid in the active site, which is framed by the structurally related helices between two repeats. Site-directed mutagenesis confirmed the importance of the residues lining the binding pocket in stabilizing the enzyme-substrate complex. Asp205 acts as a key catalytic residue and is responsible for activating a water molecule along with Asn131. Then, the hydroxyl group of the water molecule directly attacks the C2 atom of the substrate to release the halogen ion instead of forming an enzyme-substrate ester intermediate as observed in L-2-haloacid dehalogenases. The newly revealed structural and mechanistic information on D-DEX may inspire structure-based mutagenesis to engineer highly efficient haloacid dehalogenases.

Studies on structure-function relationships of acetolactate decarboxylase from Enterobacter cloacae.

Acetoin is an important bio-based platform chemical with wide applications. Among all bacterial strains, Enterobacter cloacae is a well-known acetoin producer via α-acetolactate decarboxylase (ALDC), which converts α-acetolactate into acetoin and is identified as the key enzyme in the biosynthetic pathway of acetoin. In this work, the enzyme properties of Enterobacter cloacae ALDC (E.c.-ALDC) were characterized, revealing a K m value of 12.19 mM and a k cat value of 0.96 s-1. Meanwhile, the optimum pH of E.c.-ALDC was 6.5, and the activity of E.c.-ALDC was activated by Mn2+, Ba2+, Mg2+, Zn2+ and Ca2+, while Cu2+ and Fe2+ significantly inhibited ALDC activity. More importantly, we solved and reported the first crystal structure of E.c.-ALDC at 2.4 Å resolution. The active centre consists of a zinc ion coordinated by highly conserved histidines (199, 201 and 212) and glutamates (70 and 259). However, the conserved Arg150 in E.c.-ALDC orients away from the zinc ion in the active centre of the molecule, losing contact with the zinc ion. Molecular docking of the two enantiomers of α-acetolactate, (R)-acetolactate and (S)-acetolactate allows us to further investigate the interaction networks of E.c.-ALDC with the unique conformation of Arg150. In the models, no direct contacts are observed between Arg150 and the substrates, which is unlikely to maintain the stabilization function of Arg150 in the catalytic reaction. The structure of E.c.-ALDC provides valuable information about its function, allowing a deeper understanding of the catalytic mechanism of ALDCs.