Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines

Walsh, Christopher T.; Chen, Huawei; Keating, Thomas A.; Hubbard, Brian K.; Losey, Heather C.; Luo, Lusong; Marshall, C. Gary; Miller, Deborah A.; Patel, Hiten M.

doi:10.1016/s1367-5931(00)00235-0

Cited by 284 publications

(209 citation statements)

References 48 publications

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“…R ecently, substrate specificity investigations along with specificity constant (k cat /K M ) measurements have provided significant insights into a number of biologically important enzymes [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Specificity studies of an enzyme for different natural or synthetic substrates can help determine the enzyme structure and physiological functions [1, 4 -7] as well as address the key residues involved in substrate binding and catalysis [2,8].…”

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confidence: 99%

“…The novobiocic acid noviosyl transferase activity of NovM and its specificity for various substrate analogs in designing novel coumarinbased antibiotics was recently characterized by Walsh and coworkers [3]. In addition, efforts have been made to alter enzymes by site-directed mutagenesis in order to manipulate substrate specificity [12][13][14] or enhance enzyme catalysis [15].…”

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confidence: 99%

“…Traditionally, the general method of evaluating the specificity of a given enzyme is to measure the specificity constant of a group of substrates individually followed by comparison of measured values, regardless of the assay used for measurement [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. This requires large amounts of material and considerable effort to study substrate libraries.…”

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confidence: 99%

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Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Leary

2004

J. Am. Soc. Mass Spectrom.

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The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay. (J Am Soc Mass Spectrom 2004, 15, 233-243)

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Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Leary

2004

J. Am. Soc. Mass Spectrom.

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“…The covalent thioesterification logic, used for sequestration of a fraction of the free tyrosine pool into the initial steps of antibiotic production, also yields a molecule that is thermodynamically activated to favor the lactonization and release of the newly formed 7-hydroxy-2-aminocoumarin. The versatility of the A-T didomain of NovH as a platform for the consecutive action of NovI,J,K in aminocoumarin construction is unusual and presages use of free-standing A-T didomains in other pathways routing proteinogenic amino acids such as histidine, proline, and isoleucine into secondary metabolites (25,26,29).…”

Section: Discussionmentioning

confidence: 99%

NovJ/NovK Catalyze Benzylic Oxidation of a β-Hydroxyl Tyrosyl-S-pantetheinyl Enzyme during Aminocoumarin Ring Formation in Novobiocin Biosynthesis

Pacholec

2005

Biochemistry

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The bicyclic coumarin ring in the aminocoumarin natural product antibiotics that target bacterial DNA gyrase is assembled from tyrosine by nonribosomal peptide synthetase logic. Tyrosine has previously been shown to be activated and installed as a phosphopantetheinyl thioester on the thiolation domain of NovH and then hydroxylated on the benzylic carbon by the heme protein NovI, generating -OH-Tyr-S-NovH. This aminoacyl-S-protein is the substrate for the next two orfs, Streptomyces sphaeroides NovJ and NovK, that have now been expressed in and purified from Escherichia coli as a J 2 K 2 heterotetramer. NovJ/NovK use NADP as an electron acceptor to oxidize the -OH of the tyrosyl moiety to yield the tethered -ketotyrosyl-S-NovH. The enol tautomer is the form that predominates in the subsequently cyclized aminocoumarin scaffold. The labile -ketotyrosyl thioester moiety was identified by hydrolytic release from NovH, analysis by mass spectroscopy, and comparison with a synthetic sample. We also have identified a residue in NovJ that when mutated results in a 50-fold reduction in catalytic activity.

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“…In the larger context of complex natural product biosynthesis, an early stage intermediate, such as taxadiene, provides a platform for tailoring reactions to produce the final compound. These include reduction, hydroxylation, acetylation, glycosylation, methylation, and other modifications to confer the specific molecular architecture needed for bioactivity (Olano et al 2010;Walsh et al 2001). In the case of Taxol, there are reactions dedicated to hydroxylations, oxidation, epoxidation, acetylations, benzoylations, and an amino acid modification and addition.…”

Section: Introductionmentioning

confidence: 99%

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

Jiang

Stephanopoulos

Pfeifer

2012

Appl Microbiol Biotechnol

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Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines

Cited by 284 publications

References 48 publications

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

NovJ/NovK Catalyze Benzylic Oxidation of a β-Hydroxyl Tyrosyl-S-pantetheinyl Enzyme during Aminocoumarin Ring Formation in Novobiocin Biosynthesis

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

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