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Teasing apart the Taxol pathway
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News & Comment
TRENDS in Biochemical Sciences Vol.26 No.3 March 2001
Teasing apart the Taxol pathway The diterpenoid Taxol (paclitaxel) from the yew (Taxus) species has been used successfully in the treatment of ovarian, breast and lung cancers, as well as of Kaposi’s sarcoma. The increased demand for Taxol, coupled with its limited availability from the protected Pacific yew, has had researchers scrambling for alternate sources, including synthetic and semi-synthetic pathways. Its structural complexity, however, has precluded a total synthesis suitable for large-scale production. The biosynthetic route to Taxol is no less intricate than that accomplished in the laboratory: there are a dozen enzymatic steps, including five acyltransferase reactions. Croteau and co-workers at Washington State University have already identified several enzymes in the Taxol biosynthetic pathway, including the first and third acyltransferases. Based on the abundance of naturally occurring taxoids, benzoylation at the taxane C2α-hydroxyl position was expected to be the second acylation step. Now, Walker and Croteau report the cloning from Taxus cuspidata of taxane 2α-O-benzoyltransferase, the enzyme responsible for this acylation reaction1.
The researchers tricked the plant cells into producing more Taxol (and therefore more metabolic enzymes) by incubation with methyl jasmonate, a plant signaling molecule involved in environmental stress responses. Several potential clones were isolated using transacylase-specific primers, and these clones exhibited high (64–72%) sequence similarity to previously identified enzymes in the family. Putative constructs were expressed in Escherichia coli and isolated, then evaluated for their ability to use radiolabeled benzoyl-CoA in a transferase reaction. The natural diterpenoid substrate was not available in sufficient quantities to assay the expressed protein, so 2-debenzoyl-7,13-diacetylbaccatin III was prepared semisynthetically. Upon incubation with the soluble enzyme fraction, a biosynthetic product corresponding to authentic 7,13-diacetylbaccatin III was identified. Product formation was regioselective for the 2α-hydroxyl position and dependent on a high level of ring substitution. The functional benzoyltransferase is a 440-residue protein with over 60% sequence identity to the other two acyltransferases in the Taxol
pathway, and contains the previously identified HXXXDG motif that might contribute to acyl group transfer. Taxol achieves its anticancer activity by a novel mechanism: it promotes the assembly of tubulin into microtubules, thereby disrupting mitosis. Taxol resistance is already a problem in several tumor lines, and seems due in part to changes in post-translational modifications of tubulin. Structure–function studies of Taxol have suggested that the C2 benzoate moiety is necessary for tubulin stabilization, and that substitutions at this position could yield taxol derivatives with enhanced potency. Taxane 2α-O-benzoyltransferase might provide an enzymatic means for enhanced production of Taxol and new anticancer drugs derived from this remarkable plant product.
thousands of hits, more than 70% of which are not related to the nuclear HMGs. The nomenclature of the HMG nuclear proteins has been revised to (1) facilitate interactions between various laboratories; (2) expedite literature searches; and (3) avoid confusion owing to similarity in the names of unrelated proteins. The revisions are based on the guidelines endorsed by the mouse and human gene nomenclature committees, and on consultations with the staff of both the National Center for
Biotechnology Information and the MESH Section at the National Library of Medicine in Bethesda, USA. The HMG proteins are subdivided into 3 superfamilies: HMGB (root symbol, HMGB), HMGN (root symbol, HMGN) and HMGA (root symbol, HMGA). Each HMG superfamily has a characteristic functional sequence motif. The functional motif of the HMGB family is called the ‘HMG-box’; that of the HMGN family is called the ‘nucleosomal binding domain’; and that of
1 K. Walker and R. Croteau (2000) Taxol biosynthesis: molecular cloning of a benzoyl-CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 97, 13591–13596
Rebecca W. Alexander [email protected]
Letters
Revised nomenclature for high mobility group (HMG) chromosomal proteins The high mobility group (HMG) chromosomal proteins were discovered in mammalian cells more than 30 years ago and named according to their electrophoretic mobility in polyacrylamide gels. Subsequent studies have revealed that the functional motifs characteristic of the original, canonical HMG proteins, are widespread among nuclear proteins from various organisms. A systematic way to name this group of nuclear proteins has not yet been devised and the root symbol HMG presently serves as an identifier of several proteins that are not related to nuclear HMG proteins. In fact, a literature search with the term HMG (in PubMed) gives
Table 1. Revised nomenclature for the HMG chromosomal proteinsa HMG motif protein
Functional motif
HMG-box proteins HMG-box NBD proteins NBD ATH proteins ATH
aAbbreviations:
HMG proteins Root symbol New name Old name (canonical HMGs) (canonical HMGs) HMGB HMGN HMGA
HMGB1,2,..n HMGN1,2,..n HMGA1,2,..n
HMG-1/HMG-2 HMG-14/HMG-17 HMG-I/HMG-Y/ HMG-C
ATH, AT-hook; HMG, high mobility group; NBD, nucleosome binding domain.
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Biochemical Sciences Vol.26 No.3 March 2001
Teasing apart the Taxol pathway The diterpenoid Taxol (paclitaxel) from the yew (Taxus) species has been used successfully in the treatment of ovarian, breast and lung cancers, as well as of Kaposi’s sarcoma. The increased demand for Taxol, coupled with its limited availability from the protected Pacific yew, has had researchers scrambling for alternate sources, including synthetic and semi-synthetic pathways. Its structural complexity, however, has precluded a total synthesis suitable for large-scale production. The biosynthetic route to Taxol is no less intricate than that accomplished in the laboratory: there are a dozen enzymatic steps, including five acyltransferase reactions. Croteau and co-workers at Washington State University have already identified several enzymes in the Taxol biosynthetic pathway, including the first and third acyltransferases. Based on the abundance of naturally occurring taxoids, benzoylation at the taxane C2α-hydroxyl position was expected to be the second acylation step. Now, Walker and Croteau report the cloning from Taxus cuspidata of taxane 2α-O-benzoyltransferase, the enzyme responsible for this acylation reaction1.
The researchers tricked the plant cells into producing more Taxol (and therefore more metabolic enzymes) by incubation with methyl jasmonate, a plant signaling molecule involved in environmental stress responses. Several potential clones were isolated using transacylase-specific primers, and these clones exhibited high (64–72%) sequence similarity to previously identified enzymes in the family. Putative constructs were expressed in Escherichia coli and isolated, then evaluated for their ability to use radiolabeled benzoyl-CoA in a transferase reaction. The natural diterpenoid substrate was not available in sufficient quantities to assay the expressed protein, so 2-debenzoyl-7,13-diacetylbaccatin III was prepared semisynthetically. Upon incubation with the soluble enzyme fraction, a biosynthetic product corresponding to authentic 7,13-diacetylbaccatin III was identified. Product formation was regioselective for the 2α-hydroxyl position and dependent on a high level of ring substitution. The functional benzoyltransferase is a 440-residue protein with over 60% sequence identity to the other two acyltransferases in the Taxol
pathway, and contains the previously identified HXXXDG motif that might contribute to acyl group transfer. Taxol achieves its anticancer activity by a novel mechanism: it promotes the assembly of tubulin into microtubules, thereby disrupting mitosis. Taxol resistance is already a problem in several tumor lines, and seems due in part to changes in post-translational modifications of tubulin. Structure–function studies of Taxol have suggested that the C2 benzoate moiety is necessary for tubulin stabilization, and that substitutions at this position could yield taxol derivatives with enhanced potency. Taxane 2α-O-benzoyltransferase might provide an enzymatic means for enhanced production of Taxol and new anticancer drugs derived from this remarkable plant product.
thousands of hits, more than 70% of which are not related to the nuclear HMGs. The nomenclature of the HMG nuclear proteins has been revised to (1) facilitate interactions between various laboratories; (2) expedite literature searches; and (3) avoid confusion owing to similarity in the names of unrelated proteins. The revisions are based on the guidelines endorsed by the mouse and human gene nomenclature committees, and on consultations with the staff of both the National Center for
Biotechnology Information and the MESH Section at the National Library of Medicine in Bethesda, USA. The HMG proteins are subdivided into 3 superfamilies: HMGB (root symbol, HMGB), HMGN (root symbol, HMGN) and HMGA (root symbol, HMGA). Each HMG superfamily has a characteristic functional sequence motif. The functional motif of the HMGB family is called the ‘HMG-box’; that of the HMGN family is called the ‘nucleosomal binding domain’; and that of
1 K. Walker and R. Croteau (2000) Taxol biosynthesis: molecular cloning of a benzoyl-CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 97, 13591–13596
Rebecca W. Alexander [email protected]
Letters
Revised nomenclature for high mobility group (HMG) chromosomal proteins The high mobility group (HMG) chromosomal proteins were discovered in mammalian cells more than 30 years ago and named according to their electrophoretic mobility in polyacrylamide gels. Subsequent studies have revealed that the functional motifs characteristic of the original, canonical HMG proteins, are widespread among nuclear proteins from various organisms. A systematic way to name this group of nuclear proteins has not yet been devised and the root symbol HMG presently serves as an identifier of several proteins that are not related to nuclear HMG proteins. In fact, a literature search with the term HMG (in PubMed) gives
Table 1. Revised nomenclature for the HMG chromosomal proteinsa HMG motif protein
Functional motif
HMG-box proteins HMG-box NBD proteins NBD ATH proteins ATH
aAbbreviations:
HMG proteins Root symbol New name Old name (canonical HMGs) (canonical HMGs) HMGB HMGN HMGA
HMGB1,2,..n HMGN1,2,..n HMGA1,2,..n
HMG-1/HMG-2 HMG-14/HMG-17 HMG-I/HMG-Y/ HMG-C
ATH, AT-hook; HMG, high mobility group; NBD, nucleosome binding domain.
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.