scholarly journals Algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into anN-prenyltransferase

2020 ◽  
Vol 117 (23) ◽  
pp. 12799-12805 ◽  
Author(s):  
Jonathan R. Chekan ◽  
Shaun M. K. McKinnie ◽  
Joseph P. Noel ◽  
Bradley S. Moore
Keyword(s):  
Kainic Acid ◽  
Domoic Acid ◽  
Single Amino Acid ◽  
Terpene Synthase ◽  
Binding Motif ◽  
Geranyl Diphosphate ◽  
New Family ◽  
Terpene Cyclase ◽  
Single Amino Acid Change ◽  
Domains Of Life

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for theN-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.

scholarly journals Algal Neurotoxin Biosynthesis Repurposes the Terpene Cyclase Structural Fold Into an N-prenyltransferase

2020 ◽  
Author(s):  
Jonathan R. Chekan ◽  
Shaun M. K. McKinnie ◽  
Joseph P. Noel ◽  
Bradley S. Moore
Keyword(s):  
Crystal Structure ◽  
Glutamic Acid ◽  
Kainic Acid ◽  
Domoic Acid ◽  
Marine Algae ◽  
Single Amino Acid ◽  
Binding Motif ◽  
Rational Engineering ◽  
New Family ◽  
Terpene Cyclase

AbstractPrenylation is a common biological reaction in all domains of life whereupon prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these biotransformations are structurally distinct from ubiquitous terpene cyclases that instead assemble terpene molecules via intramolecular rearrangements. Herein we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical Mg2+-binding motif. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to alter their isoprene donor specificities (geranyl versus dimethylallyl diphosphate) by a single amino acid switch. While the diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor.SignificanceDomoic acid is a neurotoxin produced by marine algae that readily bioaccumulates in shellfish and significantly impacts both human and animal life. The first committed step of the biosynthesis of domoic acid is the N-prenylation of L-glutamic acid by the enzyme DabA. By solving the crystal structure of DabA, we demonstrate that this enzyme has repurposed the common terpene cyclase fold to catalyze an extremely unusual reaction, N-prenylation of an unactivated primary amine. Application of these structural insights enabled rational engineering of two N-prenyltransferase enzymes to accept alternative prenyl donors. Ultimately, these results not only expand the scope of reactions catalyzed by a terpene cyclase family member, but will help inform future domoic acid environmental monitoring efforts.

scholarly journals Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

Genetic Analysis of Caenorhabditis elegans glp-1 Mutants Suggests Receptor Interaction or Competition

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 115-132 ◽  
Author(s):  
Anita S-R Pepper ◽  
Darrell J Killian ◽  
E Jane Albert Hubbard
Keyword(s):  
Transmembrane Domain ◽  
Limiting Factors ◽  
Single Amino Acid ◽  
Receptor Interaction ◽  
Gain Of Function ◽  
Ligand Interaction ◽  
Receptor Proteins ◽  
C Elegans ◽  
Single Amino Acid Change ◽  
Functional Defect

Abstract glp-1 encodes a member of the highly conserved LIN-12/Notch family of receptors that mediates the mitosis/meiosis decision in the C. elegans germline. We have characterized three mutations that represent a new genetic and phenotypic class of glp-1 mutants, glp-1(Pro). The glp-1(Pro) mutants display gain-of-function germline pattern defects, most notably a proximal proliferation (Pro) phenotype. Each of three glp-1(Pro) alleles encodes a single amino acid change in the extracellular part of the receptor: two in the LIN-12/Notch repeats (LNRs) and one between the LNRs and the transmembrane domain. Unlike other previously described gain-of-function mutations that affect this region of LIN-12/Notch family receptors, the genetic behavior of glp-1(Pro) alleles is not consistent with simple hypermorphic activity. Instead, the mutant phenotype is suppressed by wild-type doses of glp-1. Moreover, a trans-heterozygous combination of two highly penetrant glp-1(Pro) mutations is mutually suppressing. These results lend support to a model for a higher-order receptor complex and/or competition among receptor proteins for limiting factors that are required for proper regulation of receptor activity. Double-mutant analysis with suppressors and enhancers of lin-12 and glp-1 further suggests that the functional defect in glp-1(Pro) mutants occurs prior to or at the level of ligand interaction.

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides

FEBS Letters ◽  
2000 ◽  
Vol 470 (2) ◽  
pp. 135-138 ◽  
Author(s):  
H. Vais ◽  
S. Atkinson ◽  
N. Eldursi ◽  
A.L. Devonshire ◽  
M.S. Williamson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sodium Channel ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Pyrethroid Insecticides ◽  
Highly Sensitive ◽  
Single Amino Acid Change

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

Further characterization and determination of the single amino acid change in thetsI138reovirus thermosensitive mutant

2012 ◽  
Vol 58 (5) ◽  
pp. 589-595
Author(s):  
Guy Lemay ◽  
Martin Bisaillon
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Genetic Alterations ◽  
Biological Properties ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Gene Encoding ◽  
Viral Particles ◽  
Single Amino Acid Change

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

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