Precursor-Directed Biosynthesis of Erythromycin Analogs by an Engineered Polyketide Synthase

Jacobsen, John R.; Hutchinson, C. Richard; Cane, David E.; Khosla, Chaitan

doi:10.1126/science.277.5324.367

Cited by 278 publications

(193 citation statements)

References 46 publications

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“…1) (1)(2)(3). The genetic manipulation of macrocyclic antibiotic biosynthetic pathways, including that of erythromycin, is currently under investigation as a route for the production of novel antibiotics (4). Hydroxylations catalyzed by P450 enzymes play key roles in these biosynthetic pathways, and modification of the substrate and regiospecificity of appropriate P450 enzymes is therefore of considerable interest.…”

Section: P450 Eryfmentioning

confidence: 99%

An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates

Hong

Tschirret-Guth

Montellano

2000

Journal of Biological Chemistry

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Cytochrome P450 eryF (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450 eryF by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala 245 by a threonine enables the oxidation of alternative substrates using either H 2 O 2 or O 2 /spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11␣-, 12-, and 16␣-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450 eryF -androstenedione complex. P450 eryF is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450 eryF mutants are also attractive templates for the engineering of novel P450 activities. P450 eryF1 (CYP107A1) catalyzes the stereospecific 6(S)-hydroxylation of deoxyerythronolide B (6-DEB) in the biosynthesis of erythromycin by Saccharopolyspora erythraea ( Fig. 1) (1-3). The genetic manipulation of macrocyclic antibiotic biosynthetic pathways, including that of erythromycin, is currently under investigation as a route for the production of novel antibiotics (4). Hydroxylations catalyzed by P450 enzymes play key roles in these biosynthetic pathways, and modification of the substrate and regiospecificity of appropriate P450 enzymes is therefore of considerable interest. In an early example, targeted disruption of the gene encoding P450 eryF in S. erythraea yielded a strain that was unable to hydroxylate 6-DEB and which therefore produced 6-deoxyerythromycin (3).P450 eryF , a soluble 45-kDa protein, has been crystallized, and its structure has been determined in complexes with both the natural substrate 6-DEB and alternative ligands (5, 6). Two endogenous proteins able to provide electrons for turnover of P450 eryF have been cloned and expressed (7,8), although spinach ferredoxin and FNR function as acceptable surrogate electron donors (9). The structure of P450 eryF reveals two particularly interesting features of the enzyme. One is that the active site is much larger than the active sites of the other structurally defined bacterial P450 enzymes, as expected from the size of its macrocyclic substrate. The second is that the highly conserved threonine, Thr 252 in P450 cam (CYP101), is replaced in P450 eryF by Ala 245 (5, 6). The conserved threonine is thought to be required for dioxygen bond cleavage in the activation of molecular oxy...

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Section: P450 Eryfmentioning

confidence: 99%

An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates

Hong

Tschirret-Guth

Montellano

2000

Journal of Biological Chemistry

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“…In contrast, the role (if any) of the polypeptide moiety of the ACP is less clear. Indeed, in certain polyketide synthases and non-ribosomal peptide synthetases, the requirement of an acyl carrier protein can be by-passed by presenting the condensing enzyme with appropriate CoA-or CoA-mimetic substrates (7).…”

mentioning

confidence: 99%

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Dreier¹,

Shah²,

Khosla³

1999

Journal of Biological Chemistry

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Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA: ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-␤-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of proteinprotein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 ؎ 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [ 14 C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

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“…20 Recently, the genetic localization of specific polyketide synthase enzymes has allowed manipulation of the Streptomyces genome to provide various combinations of enzyme activities. McDaniel et al 21 and Jacobsen et al 22 have demonstrated the production of novel polyketide products following such genetic programming. As the rules governing the concerted enzyme activities leading to polyketide synthesis are better understood, production of genetically designed compounds may be possible.…”

Section: Figure 8 Inhibition Of Dnase II By Dmi-2: Effect Of Methanolmentioning

confidence: 99%

Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor

et al. 1998

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Precursor-Directed Biosynthesis of Erythromycin Analogs by an Engineered Polyketide Synthase

Cited by 278 publications

References 46 publications

An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates

An A245T Mutation Conveys on Cytochrome P450eryF the Ability to Oxidize Alternative Substrates

Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor

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