Publications by authors named "Yuanheng Cai"

17 Publications

  • Page 1 of 1

Biotin attachment domain-containing proteins mediate hydroxy fatty acid-dependent inhibition of acetyl CoA carboxylase.

Plant Physiol 2021 Jan 23. Epub 2021 Jan 23.

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

Hundreds of naturally occurring specialized fatty acids (FAs) have potential as desirable chemical feedstocks if they could be produced at large scale by crop plants; however, transgenic expression of their biosynthetic genes has generally been accompanied by dramatic reductions in oil yield. For example, expression of castor (Ricinus communis) FA hydroxylase (FAH) in the Arabidopsis thaliana FA elongation mutant fae1 resulted in a 50% reduction of FA synthesis rate that was attributed to inhibition of acetyl-CoA carboxylase (ACCase) by an undefined mechanism. Here, we tested the hypothesis that the ricinoleic acid-dependent decrease in ACCase activity is mediated by biotin attachment domain-containing (BADC) proteins. BADCs are inactive homologs of biotin carboxy carrier protein that lack a biotin cofactor and can inhibit ACCase. Arabidopsis contains three BADC genes. To reduce expression levels of BADC1 and BADC3 in fae1/FAH plants, a homozygous badc1,3/fae1/FAH line was created. The rate of FA synthesis in badc1,3/fae1/FAH seeds doubled relative to fae1/FAH, restoring it to fae1 levels, increasing both native FA and HFA accumulation. Total FA per seed, seed oil content, and seed yield per plant all increased in badc1,3/fae1/FAH, to 5.8 µg, 37%, and 162 mg, respectively, relative to 4.9 µg, 33%, and 126 mg, respectively, for fae1/FAH. Transcript levels of FA synthesis-related genes, including those encoding ACCase subunits, did not significantly differ between badc1,3/fae1/FAH and fae1/FAH. These results demonstrate that BADC1 and BADC3 mediate ricinoleic acid-dependent inhibition of FA synthesis. We propose that BADC-mediated FAS inhibition as a general mechanism that limits FA accumulation in specialized FA-accumulating seeds.
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http://dx.doi.org/10.1093/plphys/kiaa109DOI Listing
January 2021

Structural and Functional Analyses of the Transcription Repressor DgoR From Reveal a Divalent Metal-Containing D-Galactonate Binding Pocket.

Front Microbiol 2020 5;11:590330. Epub 2020 Nov 5.

Department of Microbiology and Immunology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, China.

The transcription repressor of D-galactonate metabolism, DgoR, from belongs to the FadR family of the GntR superfamily. In the presence of D-galactonate, DgoR binds to two inverted repeats overlapping the cis-acting promoter repressing the expression of genes involved in D-galactonate metabolism. To further understand the structural and molecular details of ligand and effector interactions between D-galactonate and this FadR family member, herein we solved the crystal structure of C-terminal domain of DgoR (DgoR_C), which revealed a unique divalent metal-containing substrate binding pocket. The metal ion is required for D-galactonate binding, as evidenced by the dramatically decreased affinity between D-galactonate and DgoR in the presence of EDTA, which can be reverted by the addition of Zn, Mg, and Ca. The key amino acid residues involved in the interactions between D-galactonate and DgoR were revealed by molecular docking studies and further validated with biochemical studies by site-directed mutagenesis. It was found that changes to alanine in residues R102, W181, T191, and R224 resulted in significantly decreased binding affinities for D-galactonate, as determined by EMSA and MST assays. These results suggest that the molecular modifications induced by a D-galactonate and a metal binding in the DgoR are required for DNA binding activity and consequently, transcriptional inhibition.
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http://dx.doi.org/10.3389/fmicb.2020.590330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674646PMC
November 2020

A conserved evolutionary mechanism permits Δ9 desaturation of very-long-chain fatty acyl lipids.

J Biol Chem 2020 08 11;295(32):11337-11345. Epub 2020 Jun 11.

Biochemistry & Cell Biology Department, Stony Brook University, Stony Brook, New York, USA

Δ9 fatty acyl desaturases introduce a -double bond between C9 and C10 of saturated fatty acyl chains. From the crystal structure of the mouse stearoyl-CoA desaturase (mSCD1) it was proposed that Tyr-104, a surface residue located at the distal end of the fatty acyl binding pocket plays a key role in specifying 18C selectivity. We created mSCD1-Y104G to test the hypothesis that eliminating this bulky side chain would create an opening and permit the substrate's methyl end to protrude through the enzyme into the lipid bilayer, facilitating the desaturation of very-long-chain (VLC) substrates. Consistent with this hypothesis, Y104G acquired the ability to desaturate 24C and 26C acyl-CoAs while maintaining its Δ9-regioselectivity. We also investigated two distantly related very-long-chain fatty acyl (VLCFA) desaturases from Arabidopsis, ADS1.2 and ADS1.4, which have Ala and Gly, respectively, in place of the gatekeeping Tyr found in mSCD1. Substitution of Tyr for Ala and Gly in ADS1.2 and ADS1.4, respectively, blocked their ability to desaturate VLCFAs. Further, we identified a pair of fungal desaturase homologs which contained either an Ile or a Gly at this location and showed that only the Gly-containing desaturase was capable of very-long-chain desaturation. The conserved desaturase architecture wherein a surface residue with a single bulky side chain forms the end of the substrate binding cavity predisposes them to single amino acid substitutions that enable a switch between long- and very-long-chain selectivity. The data presented here show that such changes have independently occurred multiple times during evolution.
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http://dx.doi.org/10.1074/jbc.RA120.014258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415989PMC
August 2020

Investigations into the Antibacterial Mechanism of Action of Viridicatumtoxins.

ACS Infect Dis 2020 07 4;6(7):1759-1769. Epub 2020 Jun 4.

Department of Microbiology and Immunology, School of Medicine & Holistic Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, China.

Viridicatumtoxins are a rare class of tetracycline-like antibiotics that strongly inhibit drug-resistant Gram-positive bacteria. Although reported to exhibit inhibition activity to undecaprenyl pyrophosphate synthase (UPPS), an essential enzyme in bacterial cell wall synthesis, the biological targets and mechanism of action of viridicatumtoxins, especially the drug-target interactions, remain largely unknown. In this study, the structure of UPPS (UPPS) was first determined, uncovering that UPPS can form not only a typical functional dimer but also an unexpected atypical dimer. We then observed that viridicatumtoxins A (VirA) and B (VirB) are able to bind to UPPSs of , , and in a direct and high-affinity manner as evidenced by enzyme inhibition assay, surface plasmon resonance (SPR) binding analysis, and growth inhibition assay, demonstrating that viridicatumtoxins exert antibacterial effects through UPPS binding. The key amino acid residues involved in the interactions with VirA and VirB in UPPS binding pocket were revealed by molecular docking studies, and further validated by site-directed mutagenesis. A single mutation of UPPS at D29A, N31A, and R42A can obviously increase their affinities to VirA, while a single mutation at W228A conferred significant resistance to VirA. Moreover, translation inhibition assay showed that VirA and VirB can weakly inhibit 70S ribosome. The weak inhibition of ribosome was proposed to be attributed to steric hindrance between viridicatumtoxin ring F and 70S ribosome helix 34 by molecular docking study. Our structural, biochemical, and computational investigations on the interactions of viridicatumtoxins with UPPS and 70S ribosome not only disclosed the potential biological targets of viridicatumtoxins, but also provided a theoretical basis for structural optimization to make new viridicatumtoxin derivatives with improved antimicrobial activities.
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http://dx.doi.org/10.1021/acsinfecdis.0c00031DOI Listing
July 2020

Rhodoxanthin synthase from honeysuckle; a membrane diiron enzyme catalyzes the multistep conversation of β-carotene to rhodoxanthin.

Sci Adv 2020 Apr 22;6(17):eaay9226. Epub 2020 Apr 22.

DSM Nutritional Products, 60 Westview St, Lexington, MA 02421, USA.

Rhodoxanthin is a vibrant red carotenoid found across the plant kingdom and in certain birds and fish. It is a member of the atypical retro class of carotenoids, which contain an additional double bond and a concerted shift of the conjugated double bonds relative to the more widely occurring carotenoid pigments, and whose biosynthetic origins have long remained elusive. Here, we identify LHRS ( hydroxylase rhodoxanthin synthase), a variant β-carotene hydroxylase (BCH)-type integral membrane diiron enzyme that mediates the conversion of β-carotene into rhodoxanthin. We identify residues that are critical to rhodoxanthin formation by LHRS. Substitution of only three residues converts a typical BCH into a multifunctional enzyme that mediates a multistep pathway from β-carotene to rhodoxanthin via a series of distinct oxidation steps in which the product of each step becomes the substrate for the next catalytic cycle. We propose a biosynthetic pathway from β-carotene to rhodoxanthin.
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http://dx.doi.org/10.1126/sciadv.aay9226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176425PMC
April 2020

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry.

Plant Physiol 2020 02 5;182(2):730-738. Epub 2019 Dec 5.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973

In previous work, we identified a triple mutant of the castor () stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely -9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of O labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of O and O demonstrated that both hydroxyl oxygens originate from a single molecule of O, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming -9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.
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http://dx.doi.org/10.1104/pp.19.01111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997704PMC
February 2020

Expression of a Lychee with an Enhances Cyclopropane Fatty Acid Accumulation in Camelina Seeds.

Plant Physiol 2019 07 13;180(3):1351-1361. Epub 2019 May 13.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973

Cyclopropane fatty acids (CPAs) are useful feedstocks for biofuels and bioproducts such as lubricants and biodiesel. Our goal is to identify factors that can facilitate the accumulation of CPA in seed triacylglycerol (TAG) storage oil. We hypothesized that the poor metabolism of CPA through the TAG biosynthetic network could be overcome by the addition of enzymes from species that naturally accumulate CPA in their seed oil, such as lychee (), which contains approximately 40% CPA in TAG. Our previous work on engineering CPA accumulation in crop and model plants identified a metabolic bottleneck between phosphatidylcholine (PC), the site of CPA biosynthesis, diacylglycerol (DAG), and TAG. Here, we report the cloning and heterologous expression in camelina () of a lychee (), which encodes the enzyme that catalyzes the transfer of the phosphocholine headgroup from PC to DAG. Camelina lines coexpressing and () showed up to a 50% increase of CPA in mature seed, relative to the background. Stereospecific lipid compositional analysis showed that the expression of strongly reduced the level of C18:1 substrate at PC-1 and PC--2 (i.e. the sites of CPA synthesis), while the levels of CPA increased in PC-2, DAG-1 and DAG--2, and both -1/3 and -2 positions in TAG. Taken together, these data suggest that the addition of PDCT facilitates more efficient movement of CPA from PC to DAG and establishes LcPDCT as a useful factor to combine with others to enhance CPA accumulation in plant seed oil.
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http://dx.doi.org/10.1104/pp.19.00396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752900PMC
July 2019

Two clusters of residues contribute to the activity and substrate specificity of Fm1, a bifunctional oleate and linoleate desaturase of fungal origin.

J Biol Chem 2018 12 22;293(51):19844-19853. Epub 2018 Oct 22.

From the Biochemistry and Cell Biology Department, Stony Brook University, Stony Brook, New York 11794 and

Polyunsaturated fatty acids (PUFAs) have important industrial, physiological, and nutritional properties. Plants use the sequential activities of FAD2 and FAD3 desaturases to convert 18:1Δ9 to the important PUFA 18:3Δ9,12,15, whereas the fungus 7600 uses the bifunctional desaturase Fm1 for both reactions. Here, we used a combination of sequence comparisons, structural modeling, and mutagenesis experiments to investigate Fm1's regioselectivity and identified two functionally relevant clusters of residues that contribute to Fm1 activity. We found that cluster I (Leu, Phe, and His), located near the catalytic iron ions, predominantly affects activity, whereas cluster II (Tyr, His, and Leu), located in a helix forming the entrance to the substrate-binding pocket, mainly specifies 15-desaturation. Individual or combined substitutions of cluster II residues substantially reduced 15-desaturation. The combination of F157W from cluster I with Y280L, H284V, and L287T from cluster II created an increased-activity variant that almost completely lost the ability to desaturate at C15 and acted almost exclusively as a 12-desaturase. No variants were identified in which 15-desaturation occurred in the absence of 12-desaturation. Fm1 displayed only traces of activity with C16 substrate, but several cluster I variants exhibited increased activity with both 18:1 and 16:1 substrates, converting 16:1Δ9 to 16:3Δ9,12,15, consistent with Fm1 performing sequential + 3 desaturation reactions at C12 and then C15. We propose that cluster II residues interact with the substrate headgroup when the acyl chain contains both Δ9 and Δ12 double bonds, in which case C15 becomes positioned adjacent to the di-iron site enabling a second + 3 desaturation.
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http://dx.doi.org/10.1074/jbc.RA118.005972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314118PMC
December 2018

Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil.

Plant Biotechnol J 2018 04 18;16(4):926-938. Epub 2018 Jan 18.

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA.

Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA-producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural-occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co-expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA-accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co-expression of SfLPAT with EcCPS, di-CPA-PC increased by ~50% relative to lines expressing EcCPS alone with the di-CPA-PC primarily observed in the embryonic axis and mono-CPA-PC primarily in cotyledon tissue. EcCPS-SfLPAT lines revealed a redistribution of CPA from the sn-1 to sn-2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.
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http://dx.doi.org/10.1111/pbi.12839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866947PMC
April 2018

The MYB107 Transcription Factor Positively Regulates Suberin Biosynthesis.

Plant Physiol 2017 02 13;173(2):1045-1058. Epub 2016 Dec 13.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973 (M.G., H.Y., X.Z., Y.C., G.K., C.-J.L.); and

Suberin, a lipophilic polymer deposited in the outer integument of the Arabidopsis (Arabidopsis thaliana) seed coat, represents an essential sealing component controlling water and solute movement and protecting seed from pathogenic infection. Although many genes responsible for suberin synthesis are identified, the regulatory components controlling its biosynthesis have not been definitively determined. Here, we show that the Arabidopsis MYB107 transcription factor acts as a positive regulator controlling suberin biosynthetic gene expression in the seed coat. MYB107 coexpresses with suberin biosynthetic genes in a temporal manner during seed development. Disrupting MYB107 particularly suppresses the expression of genes involved in suberin but not cutin biosynthesis, lowers seed coat suberin accumulation, alters suberin lamellar structure, and consequently renders higher seed coat permeability and susceptibility to abiotic stresses. Furthermore, MYB107 directly binds to the promoters of suberin biosynthetic genes, verifying its primary role in regulating their expression. Identifying MYB107 as a positive regulator for seed coat suberin synthesis offers a basis for discovering the potential transcriptional network behind one of the most abundant lipid-based polymers in nature.
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http://dx.doi.org/10.1104/pp.16.01614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291039PMC
February 2017

Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase.

Nat Commun 2016 06 28;7:11989. Epub 2016 Jun 28.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.

Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.
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http://dx.doi.org/10.1038/ncomms11989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931242PMC
June 2016

Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol.

J Biol Chem 2015 Oct 16;290(44):26715-24. Epub 2015 Sep 16.

From the Biological, Environmental, and Climate Sciences Department, Brookhaven National Laboratory, Upton, New York 11973

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.
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http://dx.doi.org/10.1074/jbc.M115.684217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646325PMC
October 2015

Tailoring lignin biosynthesis for efficient and sustainable biofuel production.

Plant Biotechnol J 2014 Dec 11;12(9):1154-62. Epub 2014 Sep 11.

Biosciences Department, Brookhaven Nation Laboratory, Upton, NY, USA.

Increased global interest in a bio-based economy has reinvigorated the research on the cell wall structure and composition in plants. In particular, the study of plant lignification has become a central focus, with respect to its intractability and negative impact on the utilization of the cell wall biomass for producing biofuels and bio-based chemicals. Striking progress has been achieved in the last few years both on our fundamental understanding of lignin biosynthesis, deposition and assembly, and on the interplay of lignin synthesis with the plant growth and development. With the knowledge gleaned from basic studies, researchers are now able to invent and develop elegant biotechnological strategies to sophisticatedly manipulate the quantity and structure of lignin and thus to create economically viable bioenergy feedstocks. These concerted efforts open an avenue for the commercial production of cost-competitive biofuel to meet our energy needs.
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http://dx.doi.org/10.1111/pbi.12250DOI Listing
December 2014

Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins.

Nat Commun 2014 ;5:3274

Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA.

Cytokinins are a major group of phytohormones regulating plant growth, development and stress responses. However, in contrast to the well-defined polar transport of auxins, the molecular basis of cytokinin transport is poorly understood. Here we show that an ATP-binding cassette transporter in Arabidopsis, AtABCG14, is essential for the acropetal (root to shoot) translocation of the root-synthesized cytokinins. AtABCG14 is expressed primarily in the pericycle and stelar cells of roots. Knocking out AtABCG14 strongly impairs the translocation of trans-zeatin (tZ)-type cytokinins from roots to shoots, thereby affecting the plant's growth and development. AtABCG14 localizes to the plasma membrane of transformed cells. In planta feeding of C(14) or C(13)-labelled tZ suggests that it acts as an efflux pump and its presence in the cells directly correlates with the transport of the fed cytokinin. Therefore, AtABCG14 is a transporter likely involved in the long-distance translocation of cytokinins in planta.
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http://dx.doi.org/10.1038/ncomms4274DOI Listing
October 2015

A single amino acid determines the catalytic efficiency of two alkenal double bond reductases produced by the liverwort Plagiochasma appendiculatum.

FEBS Lett 2013 Sep 13;587(18):3122-8. Epub 2013 Aug 13.

Key Laboratory of Chemical Biology of Natural Products, Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China.

Alkenal double bond reductases (DBRs) catalyze the NADPH-dependent reduction of the α,β-unsaturated double bond of many secondary metabolites. Two alkenal double bond reductase genes PaDBR1 and PaDBR2 were isolated from the liverwort species Plagiochasma appendiculatum. Recombinant PaDBR2 protein had a higher catalytic activity than PaDBR1 with respect to the reduction of the double bond present in hydroxycinnamyl aldehydes. The residue at position 56 appeared to be responsible for this difference in enzyme activity. The functionality of a C56 to Y56 mutation in PaDBR1 was similar to that of PaDBR2. Further site-directed mutagenesis and structural modeling suggested that the phenol ring stacking between this residue and the substrate was an important determinant of catalytic efficiency.
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http://dx.doi.org/10.1016/j.febslet.2013.07.051DOI Listing
September 2013

Isolation and molecular characterization of a novel D-hydantoinase from Jannaschia sp. CCS1.

FEBS J 2009 Jul 20;276(13):3575-88. Epub 2009 May 20.

Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.

Hydantoinases (HYDs) are important enzymes for industrial production of optically pure amino acids, which are widely used as precursors for various semi-synthetic antibiotics. By a process coupling genomic data mining with activity screening, a new hydantoinase, tentatively designated HYD(Js), was identified from Jannaschia sp. CCS1 and overexpressed in Escherichia coli. The specific activity of HYD(Js) on D,L-p-hydroxyphenylhydantoin as the substrate was three times higher than that of the hydantoinase originating from Burkholderia pickettii (HYD(Bp)) that is currently used in industry. The enzyme obtained was a homotetramer with a molecular mass of 253 kDa. The pH and temperature optima for HYD(Js) were 7.6 and 50 degrees C respectively, similar to those of HYD(Bp). Kinetic analysis showed that HYD(Js) has a higher k(cat) value on D,L-p-hydroxyphenylhydantoin than HYD(Bp) does. Homology modeling and substrate docking analyses of HYD(Js) and HYD(Bp) were performed, and the results revealed an enlarged substrate binding pocket in HYD(Js), which may allow better access of substrates to the catalytic centre and could account for the increased specific activity of HYD(Js). Three amino acid residues critical for HYD(Js) activity, Phe63, Leu92 and Phe150 were also identified by substrate docking and site-directed mutagenesis. Application of this high-specific activity HYD(Js) could improve the industrial production of optically pure amino acids, such as D-p-hydroxyphenylglycine. Moreover, the structural analysis also provides new insights on enzyme-substrate interaction, which shed light on engineering of hydantoinases for high catalytic activity.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07077.xDOI Listing
July 2009

Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli.

Biochem J 2007 Mar;402(3):429-37

Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.

One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. DCase (N-carbamoyl-D-amino acid amidohydrolase) is an important enzyme involved in semi-synthesis of beta-lactam antibiotics in industry. In the present study, in order to determine the amino acid sites responsible for solubility of DCase, error-prone PCR and DNA shuffling techniques were applied to randomly mutate its coding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation were isolated. Solubility tests of these and several other mutants generated by site-directed mutagenesis indicated that three amino acid residues of DCase (Ala18, Tyr30 and Lys34) are involved in its protein solubility. In silico structural modelling analyses suggest further that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on this information, multiple engineering designated mutants were constructed by site-directed mutagenesis, among them a triple mutant A18T/Y30N/K34E (named DCase-M3) could be overexpressed in E. coli and up to 80% of it was soluble. DCase-M3 was purified to homogeneity and a comparative analysis with wild-type DCase demonstrated that DCase-M3 enzyme was similar to the native DCase in terms of its kinetic and thermodynamic properties. The present study provides new insights into recombinant protein solubility in E. coli.
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http://dx.doi.org/10.1042/BJ20061457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1863561PMC
March 2007