Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase

2017 ◽  
Vol 474 (13) ◽  
pp. 2191-2202 ◽  
Author(s):  
Xin Fang ◽  
Jian-Xu Li ◽  
Jin-Quan Huang ◽  
You-Li Xiao ◽  
Peng Zhang ◽  
...  
Keyword(s):  
Metal Binding ◽  
Rational Design ◽  
Artemisia Annua ◽  
Complex Structure ◽  
Ring Closure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Sequence Alignments ◽  
Terpene Synthases ◽  
And Function

Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7 and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399, and Thr447, which play a major role in directing catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of the substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we substantially increased amorpha-4,11-diene production in a near-additive manner by engineering Thr399 and Thr447 for product release. Our results provide new insight for the rational design of enzyme activity using synthetic biology.

scholarly journals Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase

2008 ◽  
Vol 415 (2) ◽  
pp. 257-263 ◽  
Author(s):  
Heidi L. Schubert ◽  
Ruth S. Rose ◽  
Helen K. Leech ◽  
Amanda A. Brindley ◽  
Christopher P. Hill ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Active Site ◽  
Metal Binding ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
X Ray Crystallography ◽  
General Base ◽  
And Function ◽  
Protein Variants ◽  
Identical Fashion

In Bacillus megaterium, the synthesis of vitamin B12 (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD+ as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 Å (1 Å = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.

scholarly journals Genetic and Functional Analyses of PptA, a Phospho-Form Transferase Targeting Type IV Pili in Neisseria gonorrhoeae

2007 ◽  
Vol 190 (1) ◽  
pp. 387-400 ◽  
Author(s):  
Cecilia L. Næssan ◽  
Wolfgang Egge-Jacobsen ◽  
Ryan W. Heiniger ◽  
Matthew C. Wolfgang ◽  
Finn Erik Aas ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Metal Binding ◽  
Catalytic Mechanism ◽  
Phylogenetic Analyses ◽  
Site Directed Mutagenesis ◽  
Head Group ◽  
Sequence Alignments ◽  
Type Iv ◽  
Covalent Modifications ◽  
Division Cell

ABSTRACT The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae.

scholarly journals Analysis of the Topology and Active-Site Residues of WbbF, a Putative O-Polysaccharide Synthase from Salmonella enterica Serovar Borreze

2019 ◽  
Vol 202 (5) ◽  
Author(s):  
Samantha S. Wear ◽  
Brittany A. Hunt ◽  
Bradley R. Clarke ◽  
Chris Whitfield
Keyword(s):  
Salmonella Enterica ◽  
Lipid A ◽  
Critical Role ◽  
Cellulose Synthase ◽  
Site Directed Mutagenesis ◽  
Chain Extension ◽  
Active Site Residues ◽  
Content Type ◽  
Membrane Spanning ◽  
And Function

ABSTRACT Bacterial lipopolysaccharides are major components and contributors to the integrity of Gram-negative outer membranes. The more conserved lipid A-core part of this complex glycolipid is synthesized separately from the hypervariable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm prior to translocation to the outer membrane. Three different biosynthesis strategies are recognized for OPS biosynthesis, and one, the synthase-dependent pathway, is currently confined to a single example: the O:54 antigen from Salmonella enterica serovar Borreze. Synthases are complex enzymes that have the capacity to both polymerize and export bacterial polysaccharides. Although synthases like cellulose synthase are widespread, they typically polymerize a glycan without employing a lipid-linked intermediate, unlike the O:54 synthase (WbbF), which produces an undecaprenol diphosphate-linked product. This raises questions about the overall similarity between WbbF and conventional synthases. In this study, we examine the topology of WbbF, revealing four membrane-spanning helices, compared to the eight in cellulose synthase. Molecular modeling of the glycosyltransferase domain of WbbF indicates a similar architecture, and site-directed mutagenesis confirmed that residues important for catalysis and processivity in cellulose synthase are conserved in WbbF and required for its activity. These findings indicate that the glycosyltransferase mechanism of WbbF and classic synthases are likely conserved despite the use of a lipid acceptor for chain extension by WbbF. IMPORTANCE Glycosyltransferases play a critical role in the synthesis of a wide variety of bacterial polysaccharides. These include O-antigenic polysaccharides, which form the distal component of lipopolysaccharides and provide a protective barrier important for survival and host-pathogen interactions. Synthases are a subset of glycosyltransferases capable of coupled synthesis and export of glycans. Currently, the O:54 antigen of Salmonella enterica serovar Borreze involves the only example of an O-polysaccharide synthase, and its generation of a lipid-linked product differentiates it from classical synthases. Here, we explore features conserved in the O:54 enzyme and classical synthases to shed light on the structure and function of the unusual O:54 enzyme.

scholarly journals Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B14

2010 ◽  
Vol 192 (9) ◽  
pp. 2335-2345 ◽  
Author(s):  
Dylan Dodd ◽  
Shinichi Kiyonari ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue ◽  
Glycoside Hydrolase ◽  
Site Directed Mutagenesis ◽  
Rumen Bacterium ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
Sequence Alignments ◽  
Prevotella Bryantii

ABSTRACT Prevotella bryantii B14 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded β-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k cat/Km for pNP-β-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k cat/Km for pNP-β-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between β-xylosides and β-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.

Functional protein materials: beyond elastomeric and structural proteins

2019 ◽  
Vol 10 (23) ◽  
pp. 2952-2959 ◽  
Author(s):  
Nathan A. Carter ◽  
Tijana Z. Grove
Keyword(s):  
Material Properties ◽  
Rational Design ◽  
Complex Structure ◽  
Structural Proteins ◽  
Structure Property ◽  
Functional Protein ◽  
The Past ◽  
Structure Property Relationships ◽  
Biological Structures ◽  
And Function

In the past two decades researchers have shown great interest in mimicking biological structures and their complex structure–property relationships. Herein we highlight examples of hydrogels and bioelectronic materials that illustrate the rational design of material properties and function.

scholarly journals Targeting active site residues and structural anchoring positions in terpene synthases

2021 ◽  
Vol 17 ◽  
pp. 2441-2449
Author(s):  
Anwei Hou ◽  
Jeroen S Dickschat
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Terpene Synthases ◽  
Diterpene Synthase ◽  
Streptomyces Mobaraensis

The sesterterpene synthase SmTS1 from Streptomyces mobaraensis contains several unusual residues in positions that are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed, turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions.

scholarly journals Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Abanoub A. Gad ◽  
Pedram Azimzadeh ◽  
Nariman Balenga
Keyword(s):  
Rational Design ◽  
Cell Function ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Parathyroid Cell ◽  
Extracellular Loop ◽  
Sequence Alignments ◽  
Conserved Residues ◽  
Loop 2

AbstractCleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

scholarly journals Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

2021 ◽  
Author(s):  
Abanoub A. Gad ◽  
Pedram Azimzadeh ◽  
Nariman Balenga
Keyword(s):  
Rational Design ◽  
Cell Function ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Parathyroid Cell ◽  
Extracellular Loop ◽  
Sequence Alignments ◽  
Conserved Residues ◽  
Loop 2

AbstractCleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are required for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that the ECL2 mutation ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

scholarly journals Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

2021 ◽  
Author(s):  
Abanoub A. Gad ◽  
Pedram Azimzadeh ◽  
Nariman Balenga
Keyword(s):  
Rational Design ◽  
Cell Function ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Parathyroid Cell ◽  
Extracellular Loop ◽  
Sequence Alignments ◽  
Conserved Residues ◽  
Loop 2

Abstract Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are required for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that the ECL2 mutation ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

Collagen-the Ultrastructural Element of the Bone Matrix

2018 ◽  
Vol 69 (7) ◽  
pp. 1706-1709
Author(s):  
Nicoleta Dumitru ◽  
Andra Cocolos ◽  
Andra Caragheorgheopol ◽  
Constantin Dumitrache ◽  
Ovidiu Gabriel Bratu ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Bone Microarchitecture ◽  
Complex Structure ◽  
Bone Matrix ◽  
Organic Matrix ◽  
Bone Diseases ◽  
Bone Collagen ◽  
Type I ◽  
New Therapeutic Approaches ◽  
And Function

There is an increased interest and more studies highlight the fact that bone strength depends not only on bone tissue quantity, but also on its quality, which is characterized by the geometry and shape of bones, trabecular bone microarchitecture, mineral content, organic matrix and bone turnover. Fibrillar type I collagen is the major organic component of bone matrix, providing form and a stable template for mineralization. The biomedical importance of collagen as a biomaterial for medical and cosmetic purposes and the improvement of the molecular, cellular biology and analytical technologies, led to increasing interest in establishing the structure of this protein and in setting of the relationships between sequence, structure, and function. Bone collagen crosslinking chemistry and its molecular packing structure are considered to be distinct features. This unique post-translational modifications provide to the fibrillar collagen matrices properties such as tensile strength and viscoelasticity. Understanding the complex structure of bone type I collagen as well as the dynamic nature of bone tissues will help to manage new therapeutic approaches to bone diseases.

Sign in / Sign up

Export Citation Format

Share Document