國立高雄大學圖資館 |

登入

回首頁

Quantitative analysis of acyl transferase domains of modular polyketide synthases

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Quantitative analysis of acyl transferase domains of modular polyketide synthases
作者:	Liou, Grace Fang-yi.
面頁冊數:	64 p.
附註:	Adviser: Chaitan Khosla.
附註:	Source: Dissertation Abstracts International, Volume: 64-11, Section: B, page: 5641.
Contained By:	Dissertation Abstracts International64-11B.
標題:	Engineering, Chemical.
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3111748
ISBN:	0496592785

Quantitative analysis of acyl transferase domains of modular polyketide synthases
Liou, Grace Fang-yi.

Quantitative analysis of acyl transferase domains of modular polyketide synthases [electronic resource] - 64 p.

Adviser: Chaitan Khosla.

Thesis (Ph.D.)--Stanford University, 2004.

Polyketide synthases (PKSs) are a class of multifunctional enzymes that generate remarkable structural complexity and diversity by harnessing a limited repertoire of catalytic functions in a highly controlled fashion. By varying the building blocks used at every stage of chain elongation, or the extent to which the beta-ketone functionality is reduced, a great diversity of polyketide natural products are produced, with a strong track record for pharmaceutical use. The key domain in these modular proteins responsible for building block selection is the acyl transferase (AT) domain. Engineering of polyketide synthases has proved fruitful---replacing an AT domain with a homologous AT domain specific for an alternative building block produces a predictably altered polyketide product. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. In our present work, we describe a generally applicable strategy for the overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (&sim;60 kDa). Briefly, we co-expressed AT domains with the loading acyl carrier protein of the 6-deoxyerythronolide B synthase (DEBS), producing soluble proteins that could be purified to homogeneity. Using this method, we expressed methylmalonyl-CoA specific AT-2 and AT-6 of the DEBS, malonyl-CoA specific AT-2 of the rapamycin synthase (RAPS), and ethylmalonyl-CoA specific AT-5 of the tylactone synthase (TYLS). We developed two assays for measuring the intrinsic specificity of these AT domains. The first utilizes acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor. Steady-state kinetic analysis of this reaction directly probes the didomain specificity. For less active didomains, we can measure the hydrolysis rates of different acyl-CoA substrates, thereby indirectly revealing substrate preferences. We found the specificity of AT domains to be diverse. The DEBS loading didomain accepted a variety of non-carboxylated substrates, such as propionyl-, acetyl- and butyryl-CoA. On the other hand, DEBS AT-2 and AT-6, as well as RAPS AT-2, only utilized their natural substrates. Finally, TYLS AT-2 accepted ethylmalonyl-, propylmalonyl-, and allylmalonyl-CoA, but not malonyl-, methylmalonyl-, dimethylmalonyl-, or isopropylmalonyl-CoA. These studies lay the foundation for further characterization of AT domains.

ISBN: 0496592785Subjects--Topical Terms:

226989
Engineering, Chemical.

Quantitative analysis of acyl transferase domains of modular polyketide synthases
LDR:03485nmm _2200277 _450 001 162320
005 20051017073434.5
008 230606s2004 eng d
020 $a 0496592785
035 $a 00148821
035 $a 162320
040 $a UnM $c UnM
100 0 $a Liou, Grace Fang-yi. $3 227449
245 1 0 $a Quantitative analysis of acyl transferase domains of modular polyketide synthases $h [electronic resource]
300 $a 64 p.
500 $a Adviser: Chaitan Khosla.
500 $a Source: Dissertation Abstracts International, Volume: 64-11, Section: B, page: 5641.
502 $a Thesis (Ph.D.)--Stanford University, 2004.
520 # $a Polyketide synthases (PKSs) are a class of multifunctional enzymes that generate remarkable structural complexity and diversity by harnessing a limited repertoire of catalytic functions in a highly controlled fashion. By varying the building blocks used at every stage of chain elongation, or the extent to which the beta-ketone functionality is reduced, a great diversity of polyketide natural products are produced, with a strong track record for pharmaceutical use. The key domain in these modular proteins responsible for building block selection is the acyl transferase (AT) domain. Engineering of polyketide synthases has proved fruitful---replacing an AT domain with a homologous AT domain specific for an alternative building block produces a predictably altered polyketide product. Thus far, investigations into the fundamental biochemical properties of AT domains have been hampered by the inability to produce these enzymes as self-standing polypeptides. In our present work, we describe a generally applicable strategy for the overexpression and analysis of AT domains from modular PKSs as truncated didomain proteins (&sim;60 kDa). Briefly, we co-expressed AT domains with the loading acyl carrier protein of the 6-deoxyerythronolide B synthase (DEBS), producing soluble proteins that could be purified to homogeneity. Using this method, we expressed methylmalonyl-CoA specific AT-2 and AT-6 of the DEBS, malonyl-CoA specific AT-2 of the rapamycin synthase (RAPS), and ethylmalonyl-CoA specific AT-5 of the tylactone synthase (TYLS). We developed two assays for measuring the intrinsic specificity of these AT domains. The first utilizes acyl-CoA substrates as acyl group donors and N-acetylcysteamine as the thiol acceptor. Steady-state kinetic analysis of this reaction directly probes the didomain specificity. For less active didomains, we can measure the hydrolysis rates of different acyl-CoA substrates, thereby indirectly revealing substrate preferences. We found the specificity of AT domains to be diverse. The DEBS loading didomain accepted a variety of non-carboxylated substrates, such as propionyl-, acetyl- and butyryl-CoA. On the other hand, DEBS AT-2 and AT-6, as well as RAPS AT-2, only utilized their natural substrates. Finally, TYLS AT-2 accepted ethylmalonyl-, propylmalonyl-, and allylmalonyl-CoA, but not malonyl-, methylmalonyl-, dimethylmalonyl-, or isopropylmalonyl-CoA. These studies lay the foundation for further characterization of AT domains.
590 $a School code: 0212.
650 # 0 $a Engineering, Chemical. $3 226989
650 # 0 $a Engineering, Biomedical. $3 227004
690 $a 0541
690 $a 0542
710 0 # $a Stanford University. $3 212607
773 0 # $g 64-11B. $t Dissertation Abstracts International
790 $a 0212
790 1 0 $a Khosla, Chaitan, $e advisor
791 $a Ph.D.
792 $a 2004
856 4 0 $u http://libsw.nuk.edu.tw/login?url=http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3111748 $z http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3111748