scholarly journals Improved Production of Germacrene A, a Direct Precursor of ß-elemene, in Engineered Saccharomyces Cerevisiae by Expressing a Cyanobacterial Germacrene A Synthase 

2020 ◽  
Author(s):  
Weixin Zhang ◽  
Junqi Guo ◽  
Zheng Wang ◽  
Yanwei Li ◽  
Xiangfeng Meng ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Shake Flask ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Chinese Medicinal Herb ◽  
Active Site Cavity ◽  
Improved Performance ◽  
Engineered Saccharomyces Cerevisiae ◽  
Different Sources

Abstract BackgroundThe sesquiterpene germacrene A is a direct precursor of ß-elemene that is a major component of the Chinese medicinal herb Curcuma wenyujin with prominent antitumor activity. The microbial platform for germacrene A production was previously established in Saccharomyces cerevisiae using the germacrene A synthase (LTC2) of Lactuca sativa. ResultsWe evaluated the performance of LTC2 as well as nine other identified or putative germacrene A synthases from different sources for the production of germacrene A. AvGAS, a synthase of Anabaena variabilis, was found to be the most efficient in germacrene A production in yeast. AvGAS expression alone in S. cerevisiae CEN.PK2-1D already resulted in a substantial production of germacrene A while LTC2 expression did not. Further metabolic engineering the yeast using known strategies including overexpression of tHMGR1, repression of squalene synthesis pathway and decreasing farnesol synthesis, led to an 11-fold increase in germacrene A production. Site-directed mutagenesis of AvGAS revealed that while changes of several residues located within the active site cavity severely compromised germacrene A production , substitution of Phe23 located on the lateral surface with tryptophan or valine led to a 35.2% and 21.8% increase in germacrene A production, respectively. Finally, the highest production titer of germacrene A reached 309.8 mg/L in shake-flask batch culture. ConclusionsOur study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.

scholarly journals Improved production of germacrene A, a direct precursor of ß-elemene, in engineered Saccharomyces cerevisiae by expressing a cyanobacterial germacrene A synthase

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Weixin Zhang ◽  
Junqi Guo ◽  
Zheng Wang ◽  
Yanwei Li ◽  
Xiangfeng Meng ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Shake Flask ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Chinese Medicinal Herb ◽  
Active Site Cavity ◽  
Improved Performance ◽  
Engineered Saccharomyces Cerevisiae ◽  
Different Sources

Abstract Background The sesquiterpene germacrene A is a direct precursor of ß-elemene that is a major component of the Chinese medicinal herb Curcuma wenyujin with prominent antitumor activity. The microbial platform for germacrene A production was previously established in Saccharomyces cerevisiae using the germacrene A synthase (LTC2) of Lactuca sativa. Results We evaluated the performance of LTC2 (LsGAS) as well as nine other identified or putative germacrene A synthases from different sources for the production of germacrene A. AvGAS, a synthase of Anabaena variabilis, was found to be the most efficient in germacrene A production in yeast. AvGAS expression alone in S. cerevisiae CEN.PK2-1D already resulted in a substantial production of germacrene A while LTC2 expression did not. Further metabolic engineering the yeast using known strategies including overexpression of tHMGR1 and repression of squalene synthesis pathway led to an 11-fold increase in germacrene A production. Site-directed mutagenesis of AvGAS revealed that while changes of several residues located within the active site cavity severely compromised germacrene A production, substitution of Phe23 located on the lateral surface with tryptophan or valine led to a 35.2% and 21.8% increase in germacrene A production, respectively. Finally, the highest production titer of germacrene A reached 309.8 mg/L in shake-flask batch culture. Conclusions Our study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase fromLactobacillus buchneri

2017 ◽  
Vol 73 (5) ◽  
pp. 428-437 ◽  
Author(s):  
Junji Hayashi ◽  
Yuta Mutaguchi ◽  
Yume Minemura ◽  
Noriko Nakagawa ◽  
Kazunari Yoneda ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Aminobutyrate Aminotransferase ◽  
Binding Domains ◽  
Active Site Cavity ◽  
Branched Chain ◽  
Amino Acid Racemase

Crystal structures ofLactobacillus buchneriisoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5′-phosphate (PLP), in complex withN-(5′-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex withN-(5′-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of theL. buchneriisoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-∊-caprolactam racemase fromAchromobactor obaeand γ-aminobutyrate aminotransferase fromEscherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved inL. buchneriisoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine.

scholarly journals Role of the conserved amino acids of the ‘SDN’ loop (Ser130, Asp131 and Asn132) in a class A β-lactamase studied by site-directed mutagenesis

1990 ◽  
Vol 271 (2) ◽  
pp. 399-406 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
S Lepage ◽  
J Dusart ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Enzyme Stability ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
Bond Forming ◽  
Class A ◽  
Active Site Cavity

Ser130, Asp131 and Asn132 (‘SDN’) are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

scholarly journals Hydroxylation of Steroids by a Microbial Substrate-Promiscuous P450 Cytochrome (CYP105D7): Key Arginine Residues for Rational Design

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Bingbing Ma ◽  
Qianwen Wang ◽  
Haruo Ikeda ◽  
Chunfang Zhang ◽  
Lian-Hua Xu
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Cytochrome P450 Monooxygenases ◽  
Content Type ◽  
Conversion Rates ◽  
Arginine Residues

ABSTRACT Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2β- and D-ring 16β-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's kcat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids. IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.

scholarly journals The topology of the ER-resident phospholipid methyltransferase Opi3 of Saccharomyces cerevisiae is consistent with in trans catalysis

2020 ◽  
Vol 295 (8) ◽  
pp. 2473-2482 ◽  
Author(s):  
Grzegorz Pawlik ◽  
Mike F. Renne ◽  
Matthijs A. Kol ◽  
Anton I. P. M. de Kroon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Membrane Topology ◽  
Site Directed Mutagenesis ◽  
Membrane Contact Sites ◽  
Substituted Cysteine Accessibility ◽  
Cytosolic Side ◽  
In Trans ◽  
Membrane Contact ◽  
Topology Prediction

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.

Access of the substrate to the active site of squalene and oxidosqualene cyclases: comparative inhibition, site-directed mutagenesis and homology-modelling studies

2005 ◽  
Vol 33 (5) ◽  
pp. 1202-1205 ◽  
Author(s):  
S. Oliaro-Bosso ◽  
T. Schulz-Gasch ◽  
S. Taramino ◽  
M. Scaldaferri ◽  
F. Viola ◽  
...  
Keyword(s):  
Active Site ◽  
Homology Modelling ◽  
Critical Role ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Specific Substrate ◽  
Oxidosqualene Cyclase ◽  
Active Site Cavity

Substrate access to the active-site cavity of squalene-hopene cyclase from Alicyclobacillus acidocaldarious and lanosterol synthase [OSC (oxidosqualene cyclase)] from Saccharomyces cerevisiae was studied by an inhibition, mutagenesis and homology-modelling approach. Crystal structure and homology modelling indicate that both enzymes possess a narrow constriction that separates an entrance lipophilic channel from the active-site cavity. The role of the constriction as a mobile gate that permits substrate passage was investigated by experiments in which critically located Cys residues, either present in native protein or inserted by site-directed mutagenesis, were labelled with specifically designed thiol-reacting molecules. Some amino acid residues of the yeast enzyme, selected on the basis of sequence alignment and a homology model, were individually replaced by residues bearing side chains of different lengths, charges or hydrophobicities. In some of these mutants, substitution severely reduced enzymatic activity and thermal stability. Homology modelling revealed that in these mutants some critical stabilizing interactions could no longer occur. The possible critical role of entrance channel and constriction in specific substrate recognition by eukaryotic OSC is discussed.

scholarly journals Complexed Crystal Structure of Saccharomyces cerevisiae Dihydroorotase with Inhibitor 5-Fluoroorotate Reveals a New Binding Mode

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Hong-Hsiang Guan ◽  
Yen-Hua Huang ◽  
En-Shyh Lin ◽  
Chun-Jung Chen ◽  
Cheng-Yang Huang
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Structural Similarity ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Molecular Evidence ◽  
Binding Modes ◽  
Pyrimidine Nucleotides ◽  
Quenching Method

Dihydroorotase (DHOase) possesses a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHOase catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) in the third step of the pathway for the biosynthesis of pyrimidine nucleotides and is an attractive target for potential anticancer and antimalarial chemotherapy. Crystal structures of ligand-bound DHOase show that the flexible loop extends toward the active site when CA-asp is bound (loop-in mode) or moves away from the active site, facilitating the product DHO release (loop-out mode). DHOase binds the product-like inhibitor 5-fluoroorotate (5-FOA) in a similar mode to DHO. In the present study, we report the crystal structure of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

Sign in / Sign up

Export Citation Format

Share Document