Evidence that the active site in type II dehydroquinase from Streptomyces coelicolor is near the single tryptophan

ABSTRACT Type II thioesterases (TE IIs) were shown to maintain the efficiency of polyketide synthases (PKSs) by removing acyl residues blocking extension modules. However, the substrate specificity and kinetic parameters of these enzymes differ, which may have significant consequences when they are included in engineered hybrid systems for the production of novel compounds. Here we show that thioesterase ScoT associated with polyketide synthase Cpk from Streptomyces coelicolor A3(2) is able to hydrolyze acetyl, propionyl, and butyryl residues, which is consistent with its editing function. This enzyme clearly prefers propionate, in contrast to the TE IIs tested previously, and this indicates that it may have a role in control of the starter unit. We also determined activities of ScoT mutants and concluded that this enzyme is an α/β hydrolase with Ser90 and His224 in its active site.

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Theoretical Study of the Reaction Mechanism of Streptomyces coelicolor Type II Dehydroquinase

Journal of Chemical Theory and Computation ◽

10.1021/ct800480d ◽

2009 ◽

Vol 5 (5) ◽

pp. 1284-1294 ◽

Cited By ~ 10

Author(s):

L. Mattias Blomberg ◽

Martina Mangold ◽

John B. O. Mitchell ◽

Jochen Blumberger

Keyword(s):

Reaction Mechanism ◽

Theoretical Study ◽

Streptomyces Coelicolor ◽

Type Ii ◽

Type Ii Dehydroquinase

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The Structure and Mechanism of the Type II Dehydroquinase from Streptomyces coelicolor

Structure ◽

10.1016/s0969-2126(02)00747-5 ◽

2002 ◽

Vol 10 (4) ◽

pp. 493-503 ◽

Cited By ~ 57

Author(s):

Aleksander W. Roszak ◽

David A. Robinson ◽

Tino Krell ◽

Iain S. Hunter ◽

Martyn Fredrickson ◽

...

Keyword(s):

Streptomyces Coelicolor ◽

Type Ii ◽

Type Ii Dehydroquinase

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A Simple Method for the Preparation of 3-Hydroxyiminodehydroquinate, a Potent Inhibitor of Type II Dehydroquinase.

ChemInform ◽

10.1002/chin.200308187 ◽

2003 ◽

Vol 34 (8) ◽

Author(s):

Christine Le Sann ◽

Chris Abell ◽

Andrew D. Abell

Keyword(s):

Potent Inhibitor ◽

Type Ii ◽

Simple Method ◽

Type Ii Dehydroquinase

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Molecular Recognition of Citroflavonoids Naringin and Naringenin at the Active Site of the HMG-CoA Reductase and DNA Topoisomerase Type II Enzymes of Candida spp. and Ustilago maydis

Indian Journal of Microbiology ◽

10.1007/s12088-021-00980-0 ◽

2021 ◽

Author(s):

Dulce Andrade-Pavón ◽

Omar Gómez-García ◽

Lourdes Villa-Tanaca

Keyword(s):

Molecular Recognition ◽

Active Site ◽

Ustilago Maydis ◽

Type Ii ◽

Candida Spp ◽

Dna Topoisomerase ◽

Hmg Coa Reductase ◽

Coa Reductase

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Type II thioesterase ScoT is required for coelimycin production by the modular polyketide synthase Cpk of Streptomyces coelicolor A3(2).

Acta Biochimica Polonica ◽

10.18388/abp.2014_1936 ◽

2014 ◽

Vol 61 (1) ◽

Cited By ~ 6

Author(s):

Magdalena Kotowska ◽

Jarosław Ciekot ◽

Krzysztof Pawlik

Keyword(s):

Culture Medium ◽

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Acyl Chain ◽

No Production ◽

Type I ◽

Type Ii ◽

Peptide Synthetases ◽

Modular Polyketide Synthase ◽

Editing Enzyme

Type II thioesterases were shown to maintain efficiency of modular type I polyketide synthases and nonribosomal peptide synthetases by removing acyl residues blocking extension modules. We found that thioesterase ScoT from Streptomyces coelicolor A3(2) is required for the production of the yellow-pigmented coelimycin by the modular polyketide synthase Cpk. No production of coelimycin was observed in cultures of scoT disruption mutant. Polyketide production was restored upon complementation with an intact copy of the scoT gene. An enzymatic assay showed that ScoT thioesterase can hydrolyse a 12-carbon acyl chain but the activity is too low to play a role in product release from the polyketide synthase. We conclude that ScoT is an editing enzyme necessary to maintain the activity of polyketide synthase Cpk. We provide a HPLC based method to measure the amount of coelimycin P2 in a culture medium.

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LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

Nucleic Acids Research ◽

10.1093/nar/gkz443 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6369-6385

Author(s):

Jia-Yi Fan ◽

Qian Huang ◽

Quan-Quan Ji ◽

En-Duo Wang

Keyword(s):

Tertiary Structure ◽

Streptomyces Coelicolor ◽

Catalytic Efficiency ◽

Type I ◽

Type Ii ◽

Specific Domain ◽

Recognition Mechanism ◽

Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

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Resistance Genes of Aminocoumarin Producers: Two Type II Topoisomerase Genes Confer Resistance against Coumermycin A1 and Clorobiocin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.869-877.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 869-877 ◽

Cited By ~ 41

Author(s):

Elisabeth Schmutz ◽

Agnes Mühlenweg ◽

Shu-Ming Li ◽

Lutz Heide

Keyword(s):

Resistance Genes ◽

Sequence Similarity ◽

Streptomyces Coelicolor ◽

Gene Clusters ◽

Gene Products ◽

Type Ii ◽

Biosynthetic Gene Clusters ◽

B Subunit ◽

Gene Coding ◽

Similar Gene

ABSTRACT The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A1, and clorobiocin were investigated. All three clusters contained a gyrBR resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A1 clusters were found to contain an additional, similar gene, named parYR . Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrBR and likewise of parYR in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A1, suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrBR or parYR and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A1 cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A1, suggesting that these genes may be involved in antibiotic transport.

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