1hm4
From Proteopedia
(New page: 200px<br /><applet load="1hm4" size="450" color="white" frame="true" align="right" spinBox="true" caption="1hm4, resolution 3.47Å" /> '''N219L PENTALENENE SY...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:1hm4.gif|left|200px]]<br /><applet load="1hm4" size=" | + | [[Image:1hm4.gif|left|200px]]<br /><applet load="1hm4" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1hm4, resolution 3.47Å" /> | caption="1hm4, resolution 3.47Å" /> | ||
'''N219L PENTALENENE SYNTHASE'''<br /> | '''N219L PENTALENENE SYNTHASE'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F | + | Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in k(cat) and k(cat)/K(m). By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg(2+)-binding aspartate-rich domain, (80)DDLFD, resulted in reduction in the k(cat)/K(m) for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a k(cat)/K(m) which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product beta-caryophyllene (9) (9%). Finally, the F77Y mutant had a k(cat)/K(m) which was reduced by 20-fold compared to that of the wild-type synthase. |
==About this Structure== | ==About this Structure== | ||
| - | 1HM4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_sp. Streptomyces sp.]. Active as [http://en.wikipedia.org/wiki/Pentalenene_synthase Pentalenene synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.3.7 4.2.3.7] Full crystallographic information is available from [http:// | + | 1HM4 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_sp. Streptomyces sp.]. Active as [http://en.wikipedia.org/wiki/Pentalenene_synthase Pentalenene synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.3.7 4.2.3.7] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1HM4 OCA]. |
==Reference== | ==Reference== | ||
| Line 14: | Line 14: | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Streptomyces sp.]] | [[Category: Streptomyces sp.]] | ||
| - | [[Category: Cane, D | + | [[Category: Cane, D E.]] |
| - | [[Category: Christianson, D | + | [[Category: Christianson, D W.]] |
| - | [[Category: Paschall, C | + | [[Category: Paschall, C M.]] |
[[Category: Seemann, M.]] | [[Category: Seemann, M.]] | ||
[[Category: antibiotic biosynthesis]] | [[Category: antibiotic biosynthesis]] | ||
| Line 23: | Line 23: | ||
[[Category: terpene]] | [[Category: terpene]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:02:41 2008'' |
Revision as of 11:02, 21 February 2008
|
N219L PENTALENENE SYNTHASE
Overview
Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in k(cat) and k(cat)/K(m). By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg(2+)-binding aspartate-rich domain, (80)DDLFD, resulted in reduction in the k(cat)/K(m) for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a k(cat)/K(m) which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product beta-caryophyllene (9) (9%). Finally, the F77Y mutant had a k(cat)/K(m) which was reduced by 20-fold compared to that of the wild-type synthase.
About this Structure
1HM4 is a Single protein structure of sequence from Streptomyces sp.. Active as Pentalenene synthase, with EC number 4.2.3.7 Full crystallographic information is available from OCA.
Reference
Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis., Seemann M, Zhai G, de Kraker JW, Paschall CM, Christianson DW, Cane DE, J Am Chem Soc. 2002 Jul 3;124(26):7681-9. PMID:12083921
Page seeded by OCA on Thu Feb 21 13:02:41 2008
