Cloning, sequencing, overexpression in Escherichia coli, and inactivation of the valine dehydrogenase gene in the polyether antibiotic producer Streptomyces cinnamonensis

Leiser, Andreas; Birch, Ashley

doi:10.1016/0378-1119(96)00305-8

Cited by 19 publications

(12 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Isotope labeling experiments with both polyether-and macrolide-producing streptomycetes have shown that valine can be catabolized to isobutyryl-CoA, and subsequently to methylmalonyl-CoA, for antibiotic biosynthesis (23,30) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

MeaA, a Putative Coenzyme B ₁₂ -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

Zhang

Reynolds

2001

J Bacteriol

View full text Add to dashboard Cite

The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B 12 -dependent mutases. Plasmid-based expression of meaA from the ermE‫ء‬ promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.Streptomycetes produce a large number of structurally diverse polyketide antibiotics by a process similar to long-chain fatty acid biosynthesis (20). Polyketide biosynthesis, catalyzed by polyketide synthases, uses carboxylated acyl thioesters, such as malonyl-coenzyme A (CoA), methylmalonyl-CoA, or ethylmalonyl-CoA, as extender units. These precursors form the polyketide carbon backbone and side chains, as seen in the examples of rifamycin (2), erythromycin (11), and monensin (6). Malonyl-CoA and ethylmalonyl-CoA are likely derived from the carboxylation of acetyl-CoA and butyryl-CoA, respectively (16, 35), while methylmalonyl-CoA can be produced from a variety of different pathways (6). As several methylmalonyl-CoA molecules are required to build a single polyketide (six for erythromycin, seven for monensin A, and eight for rifamycin), the levels of methylmalonyl-CoA under certain conditions may represent a limiting factor in production titers. For monensin biosynthesis, either ethylmalonyl-CoA or methylmalonyl-CoA can be used at ...

show abstract

Section: Resultsmentioning

confidence: 99%

MeaA, a Putative Coenzyme B ₁₂ -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

Zhang

Reynolds

2001

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…This vector contains a temperature-sensitive Streptomyces origin of replication, resulting in its loss from host cells grown at the non-permissive temperature of 39°C (29). The vector has been used previously for gene disruptions in S. cinnamonensis (36,37). The resulting S. cinnamonensis icmA::hygB mutant was devoid of ICM activity under the usual assay conditions, providing direct evidence for a functional role of icmA in ICM activity.…”

Section: Discussionmentioning

confidence: 99%

Cloning, Sequencing, Expression, and Insertional Inactivation of the Gene for the Large Subunit of the Coenzyme B12-dependent Isobutyryl-CoA Mutase from Streptomyces cinnamonensis

Zerbe-Burkhardt¹,

Ratnatilleke²,

Philippon³

et al. 1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Purification of the coenzyme B12-dependent isobutyryl-CoA mutase (ICM) from Streptomyces cinnamonensis gave a protein of approximately 65 kDa by SDS-polyacrylamide gel electrophoresis, whose gene icmA was cloned using sequences derived from tryptic peptide fragments. The gene encodes a protein of 566 residues (62, 487 Da), with 43-44% sequence identity to the large subunit of methylmalonyl-CoA mutase (MCM) from S. cinnamonensis and Propionibacterium shermanii. Targeted disruption of the icmA gene yielded an S. cinnamonensis mutant devoid of ICM activity. The IcmA protein is approximately 160 residues shorter than the large subunit of the bacterial MCMs, corresponding to a loss of the entire C-terminal coenzyme B12 binding domain. The sequence of the (beta/alpha)8-barrel comprising residues A1-A400 in P. shermanii MCM is highly conserved in IcmA. The protein was produced in Streptomyces lividans and Escherichia coli with an N-terminal His6 tag (His6-IcmA), but after purification His6-IcmA showed no ICM activity. In the presence of coenzyme B12, protein from S. lividans and S. cinnamonensis of approximately 17 kDa by SDS-polyacrylamide gel electrophoresis could be selectively eluted with His6-IcmA from a Ni2+ affinity column. After purification, this small subunit showed no ICM activity but gave active enzyme when recombined with coenzyme B12 and IcmA or His6-IcmA.

show abstract

“…The thermostable LeuDH cloned from Bacillus stearothermophilus [11] shares considerable sequence similarities in the catalytic and coenzyme-binding domains with the enzymes acting on other amino acids, such as glutamate (GluDH) [12,13], phenylalanine (PheDH) [14,15], and valine (ValDH) [16,17] dehydrogenases. Although the overall similarities among these enzymes are not high, sequence similarities between these dehydrogenases clearly indicate the existence of an enzyme superfamily related by divergent evolution [18].…”

Section: Methodsmentioning

confidence: 99%

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Kataoka

Tanizawa

2003

Journal of Molecular Catalysis B: Enzymatic

View full text Add to dashboard Cite

The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward L-phenylalanine and phenylpyruvate with 1.6 and 7.8% of k cat values of the wild-type enzyme for the preferred substrates, L-leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.

show abstract

Cloning, sequencing, overexpression in Escherichia coli, and inactivation of the valine dehydrogenase gene in the polyether antibiotic producer Streptomyces cinnamonensis

Cited by 19 publications

References 22 publications

MeaA, a Putative Coenzyme B ₁₂ -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

MeaA, a Putative Coenzyme B ₁₂ -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

Cloning, Sequencing, Expression, and Insertional Inactivation of the Gene for the Large Subunit of the Coenzyme B12-dependent Isobutyryl-CoA Mutase from Streptomyces cinnamonensis

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Contact Info

Product

Resources

About

Cloning, sequencing, overexpression in Escherichia coli, and inactivation of the valine dehydrogenase gene in the polyether antibiotic producer Streptomyces cinnamonensis

Cited by 19 publications

References 22 publications

MeaA, a Putative Coenzyme B 12 -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

MeaA, a Putative Coenzyme B 12 -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

Cloning, Sequencing, Expression, and Insertional Inactivation of the Gene for the Large Subunit of the Coenzyme B12-dependent Isobutyryl-CoA Mutase from Streptomyces cinnamonensis

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

Contact Info

Product

Resources

About

MeaA, a Putative Coenzyme B ₁₂ -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis

MeaA, a Putative Coenzyme B ₁₂ -Dependent Mutase, Provides Methylmalonyl Coenzyme A for Monensin Biosynthesis in Streptomyces cinnamonensis