Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

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Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

Biochemistry and Cell Biology ◽

10.1139/o97-079 ◽

1997 ◽

Vol 75 (6) ◽

pp. 687-696 ◽

Cited By ~ 20

Author(s):

Tamo Fukamizo ◽

Ryszard Brzezinski

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Structure And Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Streptomyces Sp ◽

Binding Cleft ◽

And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

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Identification of Key Amino Acid Residues Determining Product Specificity of 2,3-Oxidosqualene Cyclase in Siraitia grosvenorii

Catalysts ◽

10.3390/catal8120577 ◽

2018 ◽

Vol 8 (12) ◽

pp. 577 ◽

Cited By ~ 1

Author(s):

Jing Qiao ◽

Jiushi Liu ◽

Jingjing Liao ◽

Zuliang Luo ◽

Xiaojun Ma ◽

...

Keyword(s):

Amino Acid ◽

Site Directed Mutagenesis ◽

Bioactive Molecules ◽

Amino Acid Residues ◽

Product Specificity ◽

Oxidosqualene Cyclase ◽

Siraitia Grosvenorii ◽

Initial Substrate ◽

New Strategy ◽

Key Residues

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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Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

Biochemical Journal ◽

10.1042/bj3230415 ◽

1997 ◽

Vol 323 (2) ◽

pp. 415-419 ◽

Cited By ~ 94

Author(s):

Lakshmi KASTURI ◽

Hegang CHEN ◽

Susan H. SHAKIN-ESHLEMAN

Keyword(s):

Amino Acid ◽

Amino Acid Residue ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Free Translation ◽

A Cell ◽

And Function ◽

A Site ◽

The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

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Conserved Acidic Amino Acid Residues in a Second RNA Recognition Motif Regulate Assembly and Function of TDP-43

PLoS ONE ◽

10.1371/journal.pone.0052776 ◽

2012 ◽

Vol 7 (12) ◽

pp. e52776 ◽

Cited By ~ 10

Author(s):

Akemi Shodai ◽

Akemi Ido ◽

Noriko Fujiwara ◽

Takashi Ayaki ◽

Toshifumi Morimura ◽

...

Keyword(s):

Amino Acid ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Amino Acid Residues ◽

Acidic Amino Acid ◽

Recognition Motif ◽

And Function

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Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions

Journal of Virology ◽

10.1128/jvi.79.18.11559-11568.2005 ◽

2005 ◽

Vol 79 (18) ◽

pp. 11559-11568 ◽

Cited By ~ 8

Author(s):

Chisu Song ◽

Keith Micoli ◽

Helena Bauerova ◽

Iva Pichova ◽

Eric Hunter

Keyword(s):

Amino Acid ◽

Transmembrane Protein ◽

Critical Role ◽

Cytoplasmic Domain ◽

Virus Infectivity ◽

Amino Acid Residues ◽

Glycoprotein Complex ◽

Uptake Studies ◽

Domain Block ◽

And Function

ABSTRACT Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.

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Characterization of a Pentacyclic Triterpene Acetyltransferase Involved in the Biosynthesis of Taraxasterol and ψ-Taraxasterol Acetates in Lettuce

Frontiers in Plant Science ◽

10.3389/fpls.2021.788356 ◽

2022 ◽

Vol 12 ◽

Author(s):

Han Suk Choi ◽

Jung Yeon Han ◽

Eun Ju Cheong ◽

Yong Eui Choi

Keyword(s):

Amino Acid ◽

Ectopic Expression ◽

Fatty Acyl ◽

Amino Acid Sequences ◽

Acyl Donor ◽

Amino Acid Residues ◽

Pentacyclic Triterpene ◽

Free System ◽

Oxidosqualene Cyclase ◽

Triterpene Esters

Triterpenoids exist in a free state and/or in conjugated states, such as triterpene glycosides (saponins) or triterpene esters. There is no information on the enzyme participating in the production of triterpene esters from free triterpenes. Lettuce (Lactuca sativa) contains various pentacyclic triterpene acetates (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, β-amyrin acetates, and germanicol acetate). In this study, we report a novel triterpene acetyltransferase (LsTAT1) in lettuce involved in the biosynthesis of pentacyclic triterpene acetates from free triterpenes. The deduced amino acid sequences of LsTAT1 showed a phylogenetic relationship (43% identity) with those of sterol O-acyltransferase (AtSAT1) of Arabidopsis thaliana and had catalytic amino acid residues (Asn and His) that are typically conserved in membrane-bound O-acyltransferase (MBOAT) family proteins. An analysis of LsTAT1 enzyme activity in a cell-free system revealed that the enzyme exhibited activity for the acetylation of taraxasterol, ψ-taraxasterol, β-amyrin, α-amyrin, lupeol, and taraxerol using acetyl-CoA as an acyl donor but no activity for triterpene acylation using a fatty acyl donor. Lettuce oxidosqualene cyclase (LsOSC1) is a triterpene synthase that produces ψ-taraxasterol, taraxasterol, β-amyrin and α-amyrin. The ectopic expression of both the LsOSC1 and LsTAT1 genes in yeast and tobacco could produce taraxasterol acetate, ψ-taraxasterol acetate, β-amyrin acetate, and α-amyrin acetate. However, expression of the LsTAT1 gene in tobacco was unable to induce the conversion of intrinsic sterols (campesterol, stigmasterol, and β-sitosterol) to sterol acetates. The results demonstrate that the LsTAT1 enzyme is a new class of acetyltransferase belong to the MBOAT family that have a particular role in the acetylation of pentacyclic triterpenes and are thus functionally different from sterol acyltransferase conjugating fatty acyl esters.

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Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase

Biochemical Journal ◽

10.1042/bj3600617 ◽

2001 ◽

Vol 360 (3) ◽

pp. 617-623 ◽

Cited By ~ 35

Author(s):

Qian HAN ◽

Jianmin FANG ◽

Jianyong LI

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Aspartate Aminotransferase ◽

Expression System ◽

Xanthurenic Acid ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

Kynurenine Aminotransferase ◽

E Coli ◽

Terminal Amino

The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT). KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E. coli cells. Separation of the E. coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme. N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E. coli AspAT. Recombinant AspAT (R-AspAT), homologously expressed in an E. coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (Km = 3mM; Vmax = 7.9μmol·min−1·mg−1) and 3-hydroxy-dl-kynurenine (Km = 3.7mM; Vmax = 1.25μmol·min−1·mg−1) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50–60°C and at a pH of approx. 7.0. Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (Km = 8mM; Vmax = 20.6μmol·min−1·mg−1). The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E. coli protein is AspAT.

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