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Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases.

Dunn BJ, Watts KR, Robbins T, Cane DE, Khosla C - Biochemistry (2014)

Bottom Line: The presence of the ACP had little effect on the specificity (k(cat)/K(M)) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism.Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective.Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, ‡Department of Chemistry, and ∥Department of Biochemistry, Stanford University , Stanford, California 94305, United States.

ABSTRACT
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (k(cat)/K(M)) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT- DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.

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Related in: MedlinePlus

Kinetic analysis of the carboxyacyl-CoA substratedependence ofthe transacylation reaction catalyzed by cis- and trans-AT domains. MCoA, malonyl-CoA; MMCoA, methylmalonyl-CoA;EMCoA, ethylmalonyl-CoA. (A) DEBS AT3; (B) DSZS AT; (C) KirCII. aPreviously reported data shown for comparison.11
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fig2: Kinetic analysis of the carboxyacyl-CoA substratedependence ofthe transacylation reaction catalyzed by cis- and trans-AT domains. MCoA, malonyl-CoA; MMCoA, methylmalonyl-CoA;EMCoA, ethylmalonyl-CoA. (A) DEBS AT3; (B) DSZS AT; (C) KirCII. aPreviously reported data shown for comparison.11

Mentions: To quantify the transacylation specificity of DEBS AT3, DSZS AT,and KirCII for alternative extender units, varying concentrationsof carboxyacyl-CoA substrates were used in the presence of a fixedconcentration of the cognate holo-ACP cosubstrate. As seen in Figure 2, all AT domains had at least 20-fold specificityfor their cognate substrate. Of the three, DSZS AT had the highestsubstrate specificity, with a kcat/KM for malonyl-CoA approximately 5 orders ofmagnitude higher than the kcat/KM for either methylmalonyl-CoA or ethylmalonyl-CoA.In contrast to DEBS AT3, which could not be saturated by its cognatemethylmalonyl-CoA substrate, both trans-ATs had KM values for their cognate substrates in the2–10 μM range.


Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases.

Dunn BJ, Watts KR, Robbins T, Cane DE, Khosla C - Biochemistry (2014)

Kinetic analysis of the carboxyacyl-CoA substratedependence ofthe transacylation reaction catalyzed by cis- and trans-AT domains. MCoA, malonyl-CoA; MMCoA, methylmalonyl-CoA;EMCoA, ethylmalonyl-CoA. (A) DEBS AT3; (B) DSZS AT; (C) KirCII. aPreviously reported data shown for comparison.11
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4067149&req=5

fig2: Kinetic analysis of the carboxyacyl-CoA substratedependence ofthe transacylation reaction catalyzed by cis- and trans-AT domains. MCoA, malonyl-CoA; MMCoA, methylmalonyl-CoA;EMCoA, ethylmalonyl-CoA. (A) DEBS AT3; (B) DSZS AT; (C) KirCII. aPreviously reported data shown for comparison.11
Mentions: To quantify the transacylation specificity of DEBS AT3, DSZS AT,and KirCII for alternative extender units, varying concentrationsof carboxyacyl-CoA substrates were used in the presence of a fixedconcentration of the cognate holo-ACP cosubstrate. As seen in Figure 2, all AT domains had at least 20-fold specificityfor their cognate substrate. Of the three, DSZS AT had the highestsubstrate specificity, with a kcat/KM for malonyl-CoA approximately 5 orders ofmagnitude higher than the kcat/KM for either methylmalonyl-CoA or ethylmalonyl-CoA.In contrast to DEBS AT3, which could not be saturated by its cognatemethylmalonyl-CoA substrate, both trans-ATs had KM values for their cognate substrates in the2–10 μM range.

Bottom Line: The presence of the ACP had little effect on the specificity (k(cat)/K(M)) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism.Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective.Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, ‡Department of Chemistry, and ∥Department of Biochemistry, Stanford University , Stanford, California 94305, United States.

ABSTRACT
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (k(cat)/K(M)) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT- DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.

Show MeSH
Related in: MedlinePlus