Investigation of non-classical secretion of oxalate decarboxylase in Bacillus mojavensis XH1 mediated by exopeptide YydF: Mechanism and application.

Non-classical secretory proteins are widely found in bacteria and have been extensively studied due to their important physiological roles. However, the relevant non-classical secretory mechanisms remain unclear. In this study, we found that oxalate decarboxylase (Bacm OxDC) from Bacillus mojavensis XH1 belongs to non-classical secretory proteins. Its N-terminus showed high hydrophilicity, which was different from the conventional signal peptide. The truncation test revealed that the deletion of the N-terminus affects the structure resulting in its inability to cross the cell membrane. Further studies verified that the exported peptide YydF played an important role in the secretion process of Bacm OxDC. Experimental results on the secretion mechanism indicated that Bacm OxDC bound to the exported peptide YydF and they are translocated to the cell membrane together, after which Bacm OxDC caused cell membrane relaxation for transmembrane secretion. Thereafter, three recombinant proteins were successfully secreted with certain enzymatic activity by fusing Bacm OxDC as a guide protein with various target proteins. To the best of our knowledge, this was the first time that non-classical secretion mechanism in bacteria has been analyzed. The novel discovery may provide a reference and broaden the horizons of the secretion pathway and expression regulation of proteins.

The prFMNH2-binding chaperone LpdD assists UbiD decarboxylase activation.

The UbiD enzyme family of prenylated flavin (prFMN)-dependent reversible decarboxylases is near ubiquitously present in microbes. For some UbiD family members, enzyme activation through prFMNH2 binding and subsequent oxidative maturation of the cofactor readily occurs, both in vivo in a heterologous host and through in vitro reconstitution. However, isolation of the active holo-enzyme has proven intractable for others, notably the canonical Escherichia coli UbiD. We show that E. coli heterologous expression of the small protein LpdD-associated with the UbiD-like gallate decarboxylase LpdC from Lactobacillus plantarum-unexpectedly leads to 3,4-dihydroxybenzoic acid decarboxylation whole-cell activity. This activity was shown to be linked to endogenous E. coli ubiD expression levels. The crystal structure of the purified LpdD reveals a dimeric protein with structural similarity to the eukaryotic heterodimeric proteasome assembly chaperone Pba3/4. Solution studies demonstrate that LpdD protein specifically binds to reduced prFMN species only. The addition of the LpdD-prFMNH2 complex supports reconstitution and activation of the purified E. coli apo-UbiD in vitro, leading to modest 3,4-dihydroxybenzoic acid decarboxylation. These observations suggest that LpdD acts as a prFMNH2-binding chaperone, enabling apo-UbiD activation through enhanced prFMNH2 incorporation and subsequent oxidative maturation. Hence, while a single highly conserved flavin prenyltransferase UbiX is found associated with UbiD enzymes, our observations suggest considerable diversity in UbiD maturation, ranging from robust autocatalytic to chaperone-mediated processes. Unlocking the full (de)carboxylation scope of the UbiD-enzyme family will thus require more than UbiX coexpression.

Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase.

Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on Vmax/KM of ∼ 0.5 when reaction rates of DQCA in H2O and D2O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under Vmax/KM and Vmax conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of D2O on PhdA structure support that D2O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, "closed" conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D2O and to bind DQCA more tightly, leading to the observed rate enhancement under Vmax/KM conditions.

(De)carboxylation mechanisms of heteroaromatic substrates catalyzed by prenylated FMN-dependent UbiD decarboxylases: An in-silico study.

The UbiD enzymes are proposed to catalyze reversible (de)carboxylation reaction of unsaturated carboxylic acids using prenylated flavin mononucleotide (prFMN) as a cofactor. This positions UbiD enzymes as promising candidates for converting CO2 into valuable chemicals. However, their industrial-scale biotransformation is currently constrained by low conversion rates attributed to thermodynamic limitations. To enhance the carboxylation activity of UbiD enzymes, a molecular-level understanding of the (de)carboxylation mechanisms is necessary. In this study, we investigated the reaction mechanisms of heteroaromatic substrates catalyzed by PtHmfF, PaHudA, and AnlnD enzymes using molecular dynamics (MD) simulations and free energy calculations. Our extensive mechanistic study elucidates the mechanisms involved in the formation of the initial prFMN-substrate intermediate. Specifically, we observed nucleophilic attack during decarboxylation, while carboxylation reactions involving furoic acid, pyrrole, and indole tend to favor a 1,3-dipolar cycloaddition mechanism. Furthermore, we identified proton transfer as the rate-limiting step in the carboxylation reaction. In addition, we considered the perspectives of reaction energies and electron transfer to understand the distinct mechanisms underlying decarboxylation and carboxylation. Our calculated free energies are consistent with available experimental kinetics data. Finally, we explored how different rotamers of catalytic residues influence the efficiency of the initial intermediate formation.

Enzymatic Generation of Thioaldehyde Motifs by Flavin-Dependent Cysteine Decarboxylases for Peptide Bioconjugation and Macrocyclization.

Thioaldehyde is a highly electrophilic group under aqueous conditions and can be generated via oxidative enzymatic modifications of cysteine residues in peptides and proteins. Herein, we report the installation of thioaldehyde and aldehyde groups at the C-terminus of peptides by flavin-dependent cysteine decarboxylases from the biosynthesis of ribosomally synthesized and post-translationally modified peptides. The in situ generated (thio)aldehyde is utilized as a reactive handle for peptide bioconjugation and macrocyclization.

Reactivity of Coproheme Decarboxylase with Monovinyl, Monopropionate Deuteroheme.

Coproheme decarboxylases (ChdCs) are terminal enzymes of the coproporphyrin-dependent heme biosynthetic pathway. In this reaction, two propionate groups are cleaved from the redox-active iron-containing substrate, coproheme, to form vinyl groups of the heme b product. The two decarboxylation reactions proceed sequentially, and a redox-active three-propionate porphyrin, called monovinyl, monopropionate deuteroheme (MMD), is transiently formed as an intermediate. While the reaction mechanism for the first part of the redox reaction, which is initiated by hydrogen peroxide, has been elucidated in some detail, the second part of this reaction, starting from MMD, has not been studied. Here, we report the optimization of enzymatic MMD production by ChdC and purification by reversed-phase chromatography. With the obtained MMD, we were able to study the second part of heme b formation by actinobacterial ChdC from Corynebacterium diphtheriae, starting with Compound I formation upon the addition of hydrogen peroxide. The results indicate that the second part of the decarboxylation reaction is analogous to the first part, although somewhat slower, which is explained by differences in the active site architecture and its H-bonding network. The results are discussed in terms of known kinetic and structural data and help to fill some mechanistic gaps in the overall reaction catalyzed by ChdCs.

Amino acid positions near the active site determine the reduced activity of human ACOD1 compared to murine ACOD1.

cis-Aconitate decarboxylase (ACOD1, IRG1) converts cis-aconitate to the immunomodulatory and antibacterial metabolite itaconate. Although the active site residues of human and mouse ACOD1 are identical, the mouse enzyme is about fivefold more active. Aiming to identify the cause of this difference, we mutated positions near the active site in human ACOD1 to the corresponding residues of mouse ACOD1 and measured resulting activities in vitro and in transfected cells. Interestingly, Homo sapiens is the only species with methionine instead of isoleucine at residue 154 and introduction of isoleucine at this position increased the activity of human ACOD1 1.5-fold in transfected cells and 3.5-fold in vitro. Enzyme activity of gorilla ACOD1, which is almost identical to the human enzyme but has isoleucine at residue 154, was similar to the mouse enzyme in vitro. Met154 in human ACOD1 forms a sulfur-π bond to Phe381, which is positioned to impede access of the substrate to the active site. It appears that the ACOD1 sequence has changed at position 154 during human evolution, resulting in a pronounced decrease in activity. This change might have offered a selective advantage in diseases such as cancer.

The Catalytic Mechanism of Acetoacetate Decarboxylase: A Detailed Study of Schiff Base Formation, Protonation States, and Their Impact on Catalysis.

The enzyme acetoacetate decarboxylase (AAD) has a crucial function in the process of decarboxylating the substrate acetoacetate (AA). It has been extensively studied over the years, but its exact catalytic mechanism has remained partly unsolved due to the difficulty in assessing reaction intermediates. In this study, we combine molecular dynamics and electronic structure calculations to rediscover its catalytic mechanism. Our results show that the presence of the substrate, the acetoacetate, significantly influences the electrostatic potential of the active site. Furthermore, our simulations show that the decarboxylation reaction can take place by means of a direct proton transfer instead of via an enamine intermediate, which is thought to be strictly necessary. This work provides new insights into the role of the electrostatic interactions on the catalytic activity of AAD and for the first time connects it to the catalytic mechanism of other decarboxylases.

The role of the distal cavity in carbon monoxide stabilization in the coproheme decarboxylase enzyme from C. diphtheriae.

This work focuses on the carbon monoxide adducts of the wild-type and selected variants of the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae complexed with coproheme, monovinyl monopropionyl deuteroheme (MMD), and heme b. The UV - vis and resonance Raman spectroscopies together with the molecular dynamics simulations clearly show that the wild-type coproheme-CO adduct is characterized by two CO conformers, one hydrogen-bonded to the distal H118 residue and the other showing a weak polar interaction with the distal cavity. Instead, upon conversion to heme b, i.e. after decarboxylation of propionates 2 and 4 and rotation by 90o of the porphyrin ring inside the cavity, CO probes a less polar environment. In the absence of the H118 residue, both coproheme and heme b complexes form only the non-H-bonded CO species. The unrotated MMD-CO adduct as observed in the H118F variant, confirms that decarboxylation of propionate 2 only, does not affect the heme cavity. The rupture of both the H-bonds involving propionates 2 and 4 destabilizes the porphyrin inside the cavity with the subsequent formation of a CO adduct in an open conformation. In addition, in this work we present data on CO binding to reversed heme b, obtained by hemin reconstitution of the H118A variant, and to heme d, obtained by addition of an excess of hydrogen peroxide. The results will be discussed and compared with those reported for the representatives of the firmicute clade.

Spectrophotometric and Fluorimetric High-Throughput Assays for Phenolic Acid Decarboxylase.

Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyzes the cleavage of carbon dioxide from p-coumaric-, caffeic-, and ferulic acid with high catalytic efficiency. Real-time spectroscopic assays for decarboxylase reactions remove the necessity of extensive sample workup, which is required for HPLC, mass spectrometry, gas chromatography, or NMR methods. This work presents two robust and sensitive assays based on photometry and fluorimetry that allow decarboxylation reactions to be followed with high sensitivity while avoiding product extraction and long analysis times. Optimized assay procedures were used to measure BsPAD activity in cell lysates and to determine the kinetic constants (KM and Vmax ) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

The Role of the Hydrogen Bond Network in Maintaining Heme Pocket Stability and Protein Function Specificity of C. diphtheriae Coproheme Decarboxylase.

Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H2O2, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by coproheme decarboxylase (ChdC), in a stepwise process. Decarboxylation of propionate 2 produces an intermediate that rotates by 90° inside the protein pocket, bringing propionate 4 near the catalytic tyrosine, to allow the second decarboxylation step. The active site of ChdCs is stabilized by an extensive H-bond network involving water molecules, specific amino acid residues, and the propionate groups of the porphyrin. To evaluate the role of these H-bonds in the pocket stability and enzyme functionality, we characterized, via resonance Raman and electronic absorption spectroscopies, single and double mutants of the actinobacterial pathogen Corynebacterium diphtheriae ChdC complexed with coproheme and heme b. The selective elimination of the H-bond interactions between propionates 2, 4, 6, and 7 and the polar residues of the pocket allowed us to establish the role of each H-bond in the catalytic reaction and to follow the changes in the interactions from the substrate to the product.

Negative Cooperativity in the Mechanism of Prenylated-Flavin-Dependent Ferulic Acid Decarboxylase: A Proposal for a "Two-Stroke" Decarboxylation Cycle.

Ferulic acid decarboxylase (FDC) catalyzes the reversible carboxylation of various substituted phenylacrylic acids to produce the correspondingly substituted styrenes and CO2. FDC is a member of the UbiD family of enzymes that use prenylated-FMN (prFMN) to catalyze decarboxylation reactions on aromatic rings and C-C double bonds. Although a growing number of prFMN-dependent enzymes have been identified, the mechanism of the reaction remains poorly understood. Here, we present a detailed pre-steady-state kinetic analysis of the FDC-catalyzed reaction of prFMN with both styrene and phenylacrylic acid. Based on the pattern of reactivity observed, we propose a "two-stroke" kinetic model in which negative cooperativity between the two subunits of the FDC homodimer plays an important and previously unrecognized role in catalysis. In this model, catalysis is initiated at the high-affinity active site, which reacts with phenylacrylate to yield, after decarboxylation, the covalently bound styrene-prFMN cycloadduct. In the second stage of the catalytic cycle, binding of the second substrate molecule to the low-affinity active site drives a conformational switch that interconverts the high-affinity and low-affinity active sites. This switching of affinity couples the energetically unfavorable cycloelimination of styrene from the first site with the energetically favorable cycloaddition and decarboxylation of phenylacrylate at the second site. We note as a caveat that, at this point, the complexity of the FDC kinetics leaves open other mechanistic interpretations and that further experiments will be needed to more firmly establish or refute our proposal.

Site-Directed Mutation of Salicylate Decarboxylase Gene and Mechanism of Ginkgo Acid Decarboxylation.

Ginkgo seed is an important Chinese medicine and food resource in China, but the toxicity of ginkgo acid in it limits its application. Previous studies have found that salicylic acid decarboxylase (Sdc) has a decarboxylation degradation effect on ginkgo acid. In order to improve the decarboxylation ability of Sdc to Ginkgo acid, 11 residues of the Sdc around the substrate (salicylic acid) were determined as mutation targets according to the analysis of crystal structure of Sdc (PDB ID:6JQX), from Trichosporon moniliiforme WU-0401, and a total of 30 single point mutant enzymes and one compound mutant enzyme were obtained. With Ginkgo acid C15:1 as the substrate, it was found from activity assay that Sdc-Y64T and Sdc-P191A had higher decarboxylation activity, which increased by 105.18% and 116.74% compared with that of wild type Sdc, respectively. The optimal pH for Sdc Y64T and Sdc-P191A to decarboxylate Ginkgo acid C15:1 was 5.5, which is the same as the wild type Sdc. The optimal temperature of Sdc-P191A was 50 °C, which was consistent with that of the wild type Sdc, but the optimal temperature of the mutant Sdc-Y64T was 40 °C, which was 10 °C lower than that of wild type Sdc.

Unravelling interactions between active site residues and DMAP in the initial steps of prenylated flavin mononucleotide biosynthesis catalyzed by PaUbiX.

BACKGROUND: Prenylated flavin mononucleotide (prFMN) is a recently discovered, heavily modified flavin compound. It is the only known cofactor that enables enzymatic 1,3-dipolar cycloaddition reactions. It is produced by enzymes from the UbiX family, from flavin mononucleotide and either dimethylallyl mono- or diphosphate. prFMN biosynthesis is currently reported to be initiated by protonation of the substrate by Glu140. METHODS: Computational chemistry methods are applied herein - Constant pH MD, classical MD simulations, and QM cluster optimizations. RESULTS: Glu140 competes for a single proton with Lys129 prior to prFMN biosynthesis, but it is the latter that adopted a protonated state. Once the prenyl-FMN adduct is formed, Glu140 occurs in a protonated state far more often, while the occupancy of protonated Lys129 does not change. Lys129, Glu140, and Arg122 seem to play a key role in either stabilizing or protonating DMAP phosphate group within the PaUbiX active site throughout initial steps of prFMN biosynthesis. CONCLUSIONS: The role of Lys129 in the functioning of PaUbiX is reported for the first time. Glu140 is unlikely to act as a proton donor in prFMN biosynthesis. Instead, Lys129 and Arg122 fulfil this role. Glu140 still plays a role in contributing to hydrogen-bond network. This behavior is most likely conserved throughout the UbiX family due to the structural similarity of the active sites of those proteins. SIGNIFICANCE: Mechanistic insights into a crucial biochemical process, the biosynthesis of prFMN, are provided. This study, although purely computational, extends and perfectly complements the knowledge obtained in classical laboratory experiments.

Structural insights into UbiD reversible decarboxylation.

The ubiquitous UbiX-UbiD system is associated with a wide range of microbial (de)carboxylation reactions. Recent X-ray crystallographic studies have contributed to elucidating the enigmatic mechanism underpinning the conversion of α,ß-unsaturated acids by this system. The UbiD component utilises a unique cofactor, prenylated flavin (prFMN), generated by the bespoke action of the associated UbiX flavin prenyltransferase. Structure determination of a range of UbiX/UbiD representatives has revealed a generic mode of action for both the flavin-to-prFMN metamorphosis and the (de)carboxylation. In contrast to the conserved UbiX, the UbiD superfamily is associated with a versatile substrate range. The latter is reflected in the considerable variety of UbiD quaternary structure, dynamic behaviour and active site architecture. Directed evolution of UbiD enzymes has taken advantage of this apparent malleability to generate new variants supporting in vivo hydrocarbon production. Other applications include coupling UbiD to carboxylic acid reductase to convert alkenes into α,ß-unsaturated aldehydes via enzymatic CO2 fixation.

Mechanistic Studies of a Skatole-Forming Glycyl Radical Enzyme Suggest Reaction Initiation via Hydrogen Atom Transfer.

Gut microbial decarboxylation of amino acid-derived arylacetates is a chemically challenging enzymatic transformation which generates small molecules that impact host physiology. The glycyl radical enzyme (GRE) indoleacetate decarboxylase from Olsenella uli (Ou IAD) performs the non-oxidative radical decarboxylation of indole-3-acetate (I3A) to yield skatole, a disease-associated metabolite produced in the guts of swine and ruminants. Despite the importance of IAD, our understanding of its mechanism is limited. Here, we characterize the mechanism of Ou IAD, evaluating previously proposed hypotheses of: (1) a Kolbe-type decarboxylation reaction involving an initial 1-e- oxidation of the carboxylate of I3A or (2) a hydrogen atom abstraction from the α-carbon of I3A to generate an initial carbon-centered radical. Site-directed mutagenesis, kinetic isotope effect experiments, analysis of reactions performed in D2O, and computational modeling are consistent with a mechanism involving initial hydrogen atom transfer. This finding expands the types of radical mechanisms employed by GRE decarboxylases and non-oxidative decarboxylases, more broadly. Elucidating the mechanism of IAD decarboxylation enhances our understanding of radical enzymes and may inform downstream efforts to modulate this disease-associated metabolism.

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways.

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate-binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

The Characterization of an Efficient Phenylpyruvate Decarboxylase KDC4427, Involved in 2-Phenylethanol and IAA Production from Bacterial Enterobacter sp. CGMCC 5087.

Phenylpyruvate decarboxylase (PPDC) is a crucial enzyme that plays important roles in 2-phenylethanol (2-PE) biosynthesis. In our previous study, we screened a highly efficient PPDC KDC4427 from the novel 2-PE-producing strain Enterobacter sp. CGMCC 5087. Meanwhile, its decarboxylation activity of indolylpyruvate (IPyA) was also higher than other indolylpyruvate decarboxylases (IPDCs) reported so far. In this study, KDC4427 protein was purified and characterized, and its catalytic mechanisms were analyzed by biological methods. The optimum pH and temperature of KDC4427 was pH 6.5 and 35°C, respectively. The enzyme activity was relatively stable between pH 6 and 8 and over the range of temperatures from 25°C to 45°C. KDC4427 showed the highest catalytic efficiency on phenylpyruvic acid (PPA); meanwhile, it also showed high activity for IPyA and 2-ketobutanoic acid, and it was found that KDC4427 belongs to IPDCs by phylogenetic tree analysis. The coverage of the three-dimensional structure of KDC4427 and EcIPDC from Enterobacter cloacae was 96%. Leucine 542, one of the residues in the substrate-binding pocket, is replaced by isoleucine in KDC4427 compared with EcIPDC. Site-directed mutagenesis showed that the transition from leucine to isoleucine was unlikely to make KDC4427 have high catalytic activity for PPA and IPyA; the mutants at glutamate 468 almost completely lost catalytic activities for both PPA and IPyA, indicating that this glutamate was essential for the catalytic activity. Additionally, alanine 387 plays an important role in the substrate selectivity of KDC4427. IMPORTANCE Compared with the chemical synthesis of 2-phenylethanol (2-PE) by condensation of ethylene oxide and benzene, the biological synthesis of 2-PE is a potential method to replace the traditional process. This makes biotransformation gradually become the main way to produce high-quality 2-PE. Phenylpyruvate decarboxylase (PPDC) is the critical enzyme in 2-PE biosynthesis, and it is a momentous point of penetration to increase the production of 2-PE. In this regard, KDC4427 can catalyze phenylpyruvic acid (PPA) to phenylacetaldehyde more efficiently than any other PPDC previously reported. Moreover, it has high activity of indolepyruvate decarboxylases (IPDCs), which will be a great breakthrough in the synthesis of indole-3-acetic acid (IAA). With this study, we offer insights into the KDC4427 catalytic mechanism and significantly expand the toolbox of available α-ketoacid decarboxylases for application in biosynthesis.

Evidence Supporting Substrate Channeling between Domains of Human PAICS: A Time-Course Analysis of 13C-Bicarbonate Incorporation.

Human phosphoribosylaminoimidazole carboxylase phosphoribosylaminoimdiazole succinocarboxamide synthetase (PAICS) is a dual activity enzyme catalyzing two consecutive reactions in de novo purine nucleotide synthesis. Crystallographic structures of recombinant human PAICS suggested the channeling of 4-carboxy-5-aminoimidazole-1-ribose-5'-phosphate (CAIR) between two active sites of PAICS, while a prior work of an avian PAICS suggested otherwise. Here, we present time-course mass spectrometric data supporting the channeling of CAIR between domains of recombinant human PAICS. Time-course mass spectral analysis showed that CAIR added to the bulk solution (CAIRbulk) is decarboxylated and re-carboxylated before the accumulation of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR). An experiment with 13C-bicarbonate showed that SAICAR production was proportional to re-carboxylated CAIR instead of total CAIR or CAIRbulk. This result indicates that the SAICAR synthase domain selectively uses enzyme-made CAIR over CAIRbulk, which is consistent with the channeling model. This channeling between PAICS domains may be a part of a larger channeling process in de novo purine nucleotide synthesis.