Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity

Lee, Pyung Cheon; Petri, Ralf; Mijts, Benjamin N.; Watts, Kevin T.; Schmidt‐Dannert, Claudia

doi:10.1016/j.ymben.2004.05.003

Cited by 49 publications

(34 citation statements)

References 13 publications

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“…The mutation responsible for the modified enzymatic activity was found at a position equivalent to crucial residues in other isoprenoid synthases. The use of error-prone PCR for directed evolution of the E. coli FPPS gene (ispA) resulted in GGPPS activity of proteins having novel CLD amino acyl residues (Lee et al, 2004(Lee et al, , 2005. These residues are either further upstream from the FARM than others previously identified, in helical regions adjacent to the FARM, or near the SARM.…”

Section: Discussionmentioning

confidence: 99%

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

et al. 2006

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Isoprenoids are the most diverse and abundant group of natural products. In plants, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors to many isoprenoids having essential functions. Terpenoids and sterols are derived from FPP, whereas gibberellins, carotenoids, casbenes, taxenes, and others originate from GGPP. The corresponding synthases (FPP synthase [FPPS] and GGPP synthase [GGPPS]) catalyze, respectively, the addition of two and three isopentenyl diphosphate molecules to dimethylallyl diphosphate. Maize (Zea mays L. cv B73) endosperm cDNAs encoding isoprenoid synthases were isolated by functional complementation of Escherichia coli cells carrying a bacterial gene cluster encoding all pathway enzymes needed for carotenoid biosynthesis, except for GGPPS. This approach indicated that the maize gene products were functional GGPPS enzymes. Yet, the predicted enzyme sequences revealed FPPS motifs and homology with FPPS enzymes. In vitro assays demonstrated that indeed these maize enzymes produced both FPP and GGPP and that the N-terminal sequence affected the ratio of FPP to GGPP. Their functionality in E. coli demonstrated that these maize enzymes can be coupled with a metabolon to provide isoprenoid substrates for pathway use, and suggests that enzyme bifunctionality can be harnessed. The maize cDNAs are encoded by a small gene family whose transcripts are prevalent in endosperm beginning mid development. These maize cDNAs will be valuable tools for assessing the critical structural properties determining prenyl transferase specificity and in metabolic engineering of isoprenoid pathways, especially in cereal crops.

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Section: Discussionmentioning

confidence: 99%

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

et al. 2006

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“…Cloning of genes encoding carotenoid desaturases CrtI, CrtI14, and CrtN into the constitutive expression vector pUCmod has been previously described (17,34). Cloning of genes encoding different prenyl diphosphate synthases IspA, Fgs, and CrtE into pUCmod has been previously described (16,18,34). The gene encoding the decaprenyl diphosphate synthase (Dds) from Rhodobacter sphaeroides (DSM 158) was amplified from genomic DNA with sequence-specific primers introducing an XbaI site followed by an optimized Shine-Dalgarno sequence (34) at its 5Ј end and a EcoRI site at its 3Ј end.…”

Section: Chemicalsmentioning

confidence: 99%

“…It was thought that overexpression of these shorter chain prenyl diphosphate synthases might provide more suitable substrates for the C 30 carotenoid desaturase CrtN and perhaps direct the synthesis of extended CDB systems. The prenyl diphosphate synthases selected were E. coli C 15 FPP synthase (IspA, to increase the FPP pool in E. coli) (18), Erwinia uredovora C 20 GGPP diphosphate synthase (CrtE) (34), and Aeropyrum pernix C 25 farnesylgeranyl diphosphate (FGPP) synthase (Fgs) (16). Extracts of E. coli expressing CrtE plus CrtN and Fgs plus CrtN were yellow, suggesting the formation of products with an extended CDB system.…”

Section: In Vivo Activity Of Carotenoid Desaturase Enzymes On Uqsmentioning

confidence: 99%

Biosynthesis of Ubiquinone Compounds with Conjugated Prenyl Side Chains

Lee

Salomon

Mijts

et al. 2008

Appl Environ Microbiol

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Enzymatic steps from two different biosynthetic pathways were combined in Escherichia coli, directing the synthesis of a new class of biomolecules-ubiquinones with prenyl side chains containing conjugated double bonds. This was achieved by the activity of a C 30 carotenoid desaturase, CrtN, from Staphylococcus aureus, which exhibited an inherent flexibility in substrate recognition compared to other carotenoid desaturases. By utilizing the known plasticity of E. coli's native ubiquinone biosynthesis pathway and the unusual activity of CrtN, modified ubiquinone structures with prenyl side chains containing conjugated double bonds were generated. The side chains of the new structures were confirmed to have different degrees of desaturation by mass spectrometry and nuclear magnetic resonance analysis. In vivo 14 C labeling and in vitro activity studies showed that CrtN desaturates octaprenyl diphosphates but not the ubiquinone compounds directly. Antioxidant properties of conjugated side chain ubiquinones were analyzed in an in vitro ␤-carotene-linoleate model system and were found to be higher than the corresponding unmodified ubiquinones. These results demonstrate that by combining pathway steps from different branches of biosynthetic networks, classes of compounds not observed in nature can be synthesized and structural motifs that are functionally important can be combined or enhanced.

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“…The recently reported structure of the complex of S. cerevisiae GGPS and GGPP revealed that tyrosine 107 was supplied from the other subunit [24], as is the case for I121 and V125 in type II GGPS from P. ananatis. On the other hand, Lee et al [25] reported that, in type II FPS from E. coli, glycine substitution of aspartate 115, which also exists in a-helix E, allows the enzyme to produce GGPP. The position of the aspartate residue corresponds to V125 in P. ananatis GGPS.…”

Section: Product Determination Mechanism Of Type II Ggpsmentioning

confidence: 99%

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction

Noike¹,

Kubo²,

Nakayama³

et al. 2008

The FEBS Journal

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The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short‐chain (all‐E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate‐rich motif play a primary role in the product determination mechanism. However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate‐rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate‐rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α‐helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl diphosphate synthase.

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Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity

Cited by 49 publications

References 13 publications

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

Biosynthesis of Ubiquinone Compounds with Conjugated Prenyl Side Chains

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction

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