Incremental truncation of PHA synthases results in altered product specificity

Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during the initial substrate preorganization step, oxidosqualene cyclases (OSCs) are roughly segregated into a dammarenyl cation group that predominantly catalyzes triterpenoid precursor products and a protosteryl cation group which mostly generates sterol precursor products. The mechanism of conversion between two scaffolds is not well understood. Previously, we have characterized a promiscuous OSC from Siraitia grosvenorii (SgCS) that synthesizes a novel cucurbitane-type triterpene cucurbitadienol as its main product. By integration of homology modeling, molecular docking and site-directed mutagenesis, we discover that five key amino acid residues (Asp486, Cys487, Cys565, Tyr535, and His260) may be responsible for interconversions between chair–boat–chair and chair–chair–chair conformations. The discovery of euphol, dihydrolanosterol, dihydroxyeuphol and tirucallenol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into cucurbitane-type and lanostane-type skeletons, and provide a new strategy to identify key residues determining OSC specificity.

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Iterative Truncation (ITCHY, incremental truncation for the creation of hybrid enzymes)

Encyclopedia of Genetics, Genomics, Proteomics and Informatics ◽

10.1007/978-1-4020-6754-9_8833 ◽

2008 ◽

pp. 1041-1041

Keyword(s):

The Creation ◽

Incremental Truncation ◽

Hybrid Enzymes

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PRODUCT SPECIFICITY AND REGULATION OF TWO LAURATE HYDROXYLASES IN HIGHER PLANTS

Microsomes, Drug Oxidations and Chemical Carcinogenesis ◽

10.1016/b978-0-12-187702-6.50021-9 ◽

1980 ◽

pp. 721-724 ◽

Cited By ~ 1

Author(s):

J-P. Salaün ◽

I. Benveniste ◽

D. Reichhart ◽

R. Feyereisen ◽

F. Durst

Keyword(s):

Higher Plants ◽

Product Specificity

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Preparation of Cross-Linked Enzyme Aggregates (CLEAs) of an Inulosucrase Mutant for the Enzymatic Synthesis of Inulin-Type Fructooligosaccharides

Catalysts ◽

10.3390/catal9080641 ◽

2019 ◽

Vol 9 (8) ◽

pp. 641 ◽

Cited By ~ 2

Author(s):

Thanapon Charoenwongpaiboon ◽

Rath Pichyangkura ◽

Robert A. Field ◽

Manchumas Hengsakul Prousoontorn

Keyword(s):

Enzymatic Synthesis ◽

Biochemical Characterization ◽

Lactobacillus Reuteri ◽

Soluble Enzyme ◽

Optimum Temperature ◽

Optimum Conditions ◽

Product Specificity ◽

Product Characterization ◽

Optimum Ph ◽

Probiotic Activity

Fructooligosaccharides are well-known carbohydrate molecules that exhibit good probiotic activity and are widely used as sweeteners. Inulin-type fructooligosaccharides (IFOs) can be synthesized from sucrose using inulosucrase. In this study, cross-linked enzyme aggregates (CLEAs) of Lactobacillus reuteri 121 inulosucrase (R483A-LrInu) were prepared and used as a biocatalyst for IFOs production. Under optimum conditions, R483A-LrInu CLEAs retained 42% of original inulosucrase activity. Biochemical characterization demonstrated that the optimum pH of inulosucrase changed from 5 to 4 after immobilization, while the optimum temperature was unchanged. Furthermore, the pH stability and thermostability of the R483A-LrInu CLEAs was significantly improved. IFOs product characterization indicated that the product specificity of the enzyme was impacted by CLEA generation, producing a narrower range of IFOs than the soluble enzyme. In addition, the R483A-LrInu CLEAs showed operational stability in the batch synthesis of IFOs.

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Implications of the mutation S164A on Bacillus subtilis levansucrase product specificity and insights into protein interactions acting upon levan synthesis

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2020.06.114 ◽

2020 ◽

Vol 161 ◽

pp. 898-908 ◽

Cited By ~ 1

Author(s):

Maria Elena Ortiz-Soto ◽

Jaime Ricardo Porras-Domínguez ◽

María Elena Rodríguez-Alegría ◽

Luis Alberto Morales-Moreno ◽

Adelaida Díaz-Vilchis ◽

...

Keyword(s):

Bacillus Subtilis ◽

Protein Interactions ◽

Product Specificity

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A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry

Biomolecules ◽

10.3390/biom10010118 ◽

2020 ◽

Vol 10 (1) ◽

pp. 118 ◽

Cited By ~ 2

Author(s):

Magdalena Topolska ◽

Fernando Martínez-Montañés ◽

Christer S. Ejsing

Keyword(s):

Mass Spectrometry ◽

Fatty Acid ◽

High Resolution ◽

Fatty Acid Synthase ◽

De Novo ◽

High Resolution Mass Spectrometry ◽

Enzymatic Process ◽

Product Specificity ◽

High Resolution Mass ◽

Resolution Mass

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of de novo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.

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Induction and Transcription Studies of the Dextransucrase Gene in Leuconostoc mesenteroides NRRL B-512F

Applied and Environmental Microbiology ◽

10.1128/aem.65.12.5504-5509.1999 ◽

1999 ◽

Vol 65 (12) ◽

pp. 5504-5509 ◽

Cited By ~ 31

Author(s):

M. Quirasco ◽

A. López-Munguía ◽

M. Remaud-Simeon ◽

P. Monsan ◽

A. Farrés

Keyword(s):

High Performance ◽

Leuconostoc Mesenteroides ◽

Carbon Sources ◽

Promoter Sequence ◽

Culture Conditions ◽

Start Codon ◽

Reaction Products ◽

Product Specificity ◽

First Time

ABSTRACT Dextransucrase production by Leuconostoc mesenteroidesNRRL B-512F in media containing carbon sources other than sucrose is reported for the first time. Dextransucrases were analyzed by gel electrophoresis and by an in situ activity assay. Their polymers and acceptor reaction products were also compared by 13C nuclear magnetic resonance and high-performance liquid chromatography techniques, respectively. From these analyses, it was found that, independently of the carbon source, L. mesenteroides NRRL B-512F produced dextransucrases of the same size and product specificity. The 5′ ends of dextransucrase mRNAs isolated from cells grown under different culture conditions were identical. Based on this evidence, we conclude that dextransucrases obtained from cells grown on the various carbon sources result from the transcription of the same gene. The control of expression occurs at this level. The low dextransucrase yields from cultures in d-glucose ord-fructose and the enhancement of dextransucrase gene transcription in the presence of sucrose suggest that an activating phenomenon may be involved in the expression mechanism. Dextransucrase mRNA has a size of approximately 4.8 kb, indicating that the gene is located in a monocistronic operon. The transcription start point was localized 34 bp upstream from the ATG start codon. The −10 and −35 sequences found, TATAAT and TTTACA, were highly homologous to the only glycosyltransferase promoter sequence reported for lactic acid bacteria.

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Trehalulose Synthase Native and Carbohydrate Complexed Structures Provide Insights into Sucrose Isomerization

Journal of Biological Chemistry ◽

10.1074/jbc.m704515200 ◽

2007 ◽

Vol 282 (38) ◽

pp. 28126-28136 ◽

Cited By ~ 56

Author(s):

Stéphanie Ravaud ◽

Xavier Robert ◽

Hildegard Watzlawick ◽

Richard Haser ◽

Ralf Mattes ◽

...

Keyword(s):

Substrate Recognition ◽

Three Dimensional ◽

Enzyme Reaction ◽

Structural Data ◽

Side Reactions ◽

Binding Modes ◽

Product Specificity ◽

Reaction Specificity ◽

First Time ◽

Enzyme Targeting

Various diseases related to the overconsumption of sugar make a growing need for sugar substitutes. Because sucrose is an inexpensive and readily available d-glucose donor, the industrial potential for enzymatic synthesis of the sucrose isomers trehalulose and/or isomaltulose from sucrose is large. The product specificity of sucrose isomerases that catalyze this reaction depends essentially on the possibility for tautomerization of sucrose, which is required for trehalulose formation. For optimal use of the enzyme, targeting controlled synthesis of these functional isomers, it is necessary to minimize the side reactions. This requires an extensive analysis of substrate binding modes and of the specificity-determining sites in the structure. The 1.6-2.2-Å resolution three-dimensional structures of native and mutant complexes of a trehalulose synthase from Pseudomonas mesoacidophila MX-45 mimic successive states of the enzyme reaction. Combined with mutagenesis studies they give for the first time thorough insights into substrate recognition and processing and reaction specificities of these enzymes. Among the important outcomes of this study is the revelation of an aromatic clamp defined by Phe256 and Phe280 playing an essential role in substrate recognition and in controlling the reaction specificity, which is further supported by mutagenesis studies. Furthermore, this study highlights essential residues for binding the glucosyl and fructosyl moieties. The introduction of subtle changes informed by comparative three-dimensional structural data observed within our study can lead to fundamental modifications in the mode of action of sucrose isomerases and hence provide a template for industrial catalysts.

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