Substrate selectivity of an isolated enoyl reductase catalytic domain from an iterative highly reducing fungal polyketide synthase reveals key components of programming

The complete stereochemical course and substrate selectivity of the enoyl reductase domain from the fungal polyketide synthase squalestatin tetraketide synthase (SQTKS) have been determined.

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Role of the enoyl reductase domain in the regulation of fatty acid synthase activity by interdomain interaction.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68854-x ◽

1981 ◽

Vol 256 (16) ◽

pp. 8379-8383

Author(s):

A.J. Poulose ◽

P.E. Kolattukudy

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Enoyl Reductase ◽

Interdomain Interaction ◽

Reductase Domain

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The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production

Applied and Environmental Microbiology ◽

10.1128/aem.00963-05 ◽

2006 ◽

Vol 72 (6) ◽

pp. 3924-3932 ◽

Cited By ~ 76

Author(s):

Erik Lysï¿½e ◽

Sonja S. Klemsdal ◽

Karen R. Bone ◽

Rasmus J. N. Frandsen ◽

Thomas Johansen ◽

...

Keyword(s):

Fusarium Graminearum ◽

High Performance ◽

Polyketide Synthase ◽

Wild Type ◽

Root Infection ◽

Enoyl Reductase ◽

Transformation Protocol ◽

Chromatography Analysis ◽

In The Wild ◽

High Level

ABSTRACT Zearalenones are produced by several Fusarium species and can cause reproductive problems in animals. Some aurofusarin mutants of Fusarium pseudograminearum produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the Fusarium graminearum database was carried out for these mutants. PKS4 was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An Agrobacterium tumefaciens-mediated transformation protocol was used to replace the central part of the PKS4 gene with a hygB resistance gene through double homologous recombination in an F. graminearum strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of PKS4. High-performance liquid chromatography analysis showed that the PKS4 replacement mutant did not produce ZON. Thus, PKS4 encodes an enzyme required for the production of ZON in F. graminearum. Barley root infection studies revealed no alteration in the pathogenicity of the PKS4 mutant compared to the pathogenicity of the wild type. The expression of PKS13, which is located in the same cluster as PKS4, decreased dramatically in the mutant, while transcription of PKS4 was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of PKS4 and that the PKS4-encoded protein or its product stimulates expression of PKS13. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others.

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The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902211116 ◽

2019 ◽

Vol 116 (41) ◽

pp. 20404-20410 ◽

Cited By ~ 7

Author(s):

Amy J. Fernandez ◽

Earnest James Paul Daniel ◽

Sai Pooja Mahajan ◽

Jeffrey J. Gray ◽

Thomas A. Gerken ◽

...

Keyword(s):

Colorectal Cancer ◽

Molecular Basis ◽

Catalytic Domain ◽

Binding Pocket ◽

Substrate Selectivity ◽

Peptide Substrate ◽

X Ray ◽

Biochemical Studies ◽

Cancer Mutations ◽

Insight Into

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

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Reengineering the programming of a functional domain of an iterative highly reducing polyketide synthase

RSC Advances ◽

10.1039/d0ra04026f ◽

2020 ◽

Vol 10 (31) ◽

pp. 18469-18476

Author(s):

Oliver Piech ◽

Russell J. Cox

Keyword(s):

Polyketide Synthase ◽

Functional Domain ◽

Enoyl Reductase ◽

Directed Mutation ◽

First Time ◽

Intrinsic Program

Site-directed mutation of the enoyl reductase (ER) component of an iterative highly-reducing polyketide synthase was achieved for the first time, expanding its intrinsic program.

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Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-016-2152-2 ◽

2016 ◽

Vol 180 (6) ◽

pp. 1056-1075 ◽

Cited By ~ 1

Author(s):

Edgar Omar Mendoza-Llerenas ◽

David Javier Pérez ◽

Zeferino Gómez-Sandoval ◽

Pilar Escalante-Minakata ◽

Vrani Ibarra-Junquera ◽

...

Keyword(s):

Lactobacillus Plantarum ◽

Catalytic Domain ◽

Substrate Selectivity

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In vitro kinetic study of the squalestatin tetraketide synthase dehydratase reveals the stereochemical course of a fungal highly reducing polyketide synthase

Chemical Communications ◽

10.1039/c6cc10172k ◽

2017 ◽

Vol 53 (10) ◽

pp. 1727-1730 ◽

Cited By ~ 11

Author(s):

Emma Liddle ◽

Alan Scott ◽

Li-Chen Han ◽

David Ivison ◽

Thomas J. Simpson ◽

...

Keyword(s):

Fatty Acid ◽

Kinetic Study ◽

Fatty Acid Synthase ◽

Polyketide Synthase ◽

Substrate Selectivity ◽

Dh Domain

The substrate selectivity of the isolated dehydratase (DH) domain of a fungal highly-reducing polyketide synthase is closely related to that of mammalian fatty acid synthase.

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Nostophycin Biosynthesis Is Directed by a Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase in the Toxic Cyanobacterium Nostoc sp. Strain 152

Applied and Environmental Microbiology ◽

10.1128/aem.05993-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 8034-8040 ◽

Cited By ~ 22

Author(s):

David P. Fewer ◽

Julia Österholm ◽

Leo Rouhiainen ◽

Jouni Jokela ◽

Matti Wahlsten ◽

...

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Catalytic Domain ◽

Phylogenetic Analyses ◽

Biosynthetic Gene Cluster ◽

Nonribosomal Peptide Synthetase ◽

Open Reading Frames ◽

Nonribosomal Peptide ◽

Peptide Synthetase ◽

Biochemical Analyses

ABSTRACTCyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. Thenpngene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.

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