Irina Koryakina
North Carolina State University
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Featured researches published by Irina Koryakina.
ACS Chemical Biology | 2013
Irina Koryakina; John B. McArthur; Shan M. Randall; Matthew M. Draelos; Ewa Maria Musiol; David C. Muddiman; Tilmann Weber; Gavin J. Williams
Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides.
ChemBioChem | 2011
Irina Koryakina; Gavin J. Williams
Tailoring guide: We have used structure-guided saturation mutagenesis followed by colorimetric screening to identify mutant malonyl-CoA synthetases with altered substrate specificity. One particular mutant displayed a 240-fold shift in specificity (see graphic). These mutant enzymes will be useful tools for providing extender units to probe the activity of polyketide synthases.
ACS Chemical Biology | 2017
Irina Koryakina; Christian M. Kasey; John B. McArthur; Andrew N. Lowell; Joseph A. Chemler; Shasha Li; Douglas A. Hansen; David H. Sherman; Gavin J. Williams
Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.
Journal of Biomolecular Screening | 2011
Irina Koryakina; Jessica Neville; Koichi Nonaka; Steven G. Van Lanen; Gavin J. Williams
In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.
Rapid Communications in Mass Spectrometry | 2014
Shan M. Randall; Irina Koryakina; Gavin J. Williams; David C. Muddiman
RATIONALE Site occupancy measurements using liquid chromatography/mass spectrometry (LC/MS) are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases. METHODS Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of NPSA. RESULTS A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied. CONCLUSIONS Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements.
Rapid Communications in Mass Spectrometry | 2014
Shan M. Randall; Irina Koryakina; Gavin J. Williams; David C. Muddiman
RATIONALE Site occupancy measurements using liquid chromatography/mass spectrometry (LC/MS) are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases. METHODS Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of NPSA. RESULTS A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied. CONCLUSIONS Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements.
Rapid Communications in Mass Spectrometry | 2014
Shan M. Randall; Irina Koryakina; Gavin J. Williams; David C. Muddiman
RATIONALE Site occupancy measurements using liquid chromatography/mass spectrometry (LC/MS) are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases. METHODS Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of NPSA. RESULTS A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied. CONCLUSIONS Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements.
Organic and Biomolecular Chemistry | 2013
Irina Koryakina; John B. McArthur; Matthew M. Draelos; Gavin J. Williams
ACS Synthetic Biology | 2017
Ewa Maria Musiol-Kroll; Florian Zubeil; Thomas Schafhauser; Thomas Härtner; Andreas Kulik; John B. McArthur; Irina Koryakina; Wolfgang Wohlleben; Stephanie Grond; Gavin J. Williams; Sang Yup Lee; Tilmann Weber
Archive | 2013
Gavin J. Williams; Irina Koryakina; John B. McArthur; Matthew M. Draelos; Shan Randal; David Muddimanl