Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase) from Streptomyces griseochromogenes, the first prokaryotic UDP-glucuronosyltransferase

Gould, Steven J.; Guo, Jincan

doi:10.1128/jb.176.5.1282-1286.1994

Cited by 20 publications

(15 citation statements)

References 33 publications

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“…In addition to the structural resemblance of MIL and blasticidin S (Scheme ), fermentation and biochemical studies also support related biosynthetic pathways. Similar to the findings with MIL, yields of blasticidin S increased nearly two‐fold when free cytosine was added to cultures of the producing organism 20. More recently, BlsM was characterized from the blasticidin S biosynthetic pathway as a CMP hydrolase, and was shown to be responsible for generating free cytosine from CMP,21 analogous to the conversion of HMCMP to HMC.…”

Section: Introductionsupporting

confidence: 67%

The Mildiomycin Biosynthesis: Initial Steps for Sequential Generation of 5‐Hydroxymethylcytidine 5′‐Monophosphate and 5‐Hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

et al. 2008

ChemBioChem

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Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. We have cloned the MIL biosynthetic gene cluster in Streptoverticillum rimofaciens ZJU5119 and shown that this organism also produces the related antifungal compound, deshydroxymethyl mildiomycin (dHM-MIL). A cosmid genomic library was screened for a putative nucleotide hydrolase gene that is related to blsM from the blasticidin S cluster. Six cosmids were identified that contained a 3.5 kb DNA fragment that harbors a homologue of blsM. The sequence of the fragment revealed two open-reading frames that are likely to function in MIL formation: milA is a CMP hydroxymethylase gene and milB is the homologue of the CMP hydrolase gene blsM. Insertional disruption of milA abolished the production of MIL but not dHM-MIL, whereas a milB knockout strain did not produce either of the peptidyl nucleosides. Recombinant MilA was produced in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP, but it did not accept cytosine or dCMP as a substrate. MilB was also expressed and purified from E. coli and shown to efficiently hydrolyze both hydroxymethyl-CMP (HMCMP) and could accept CMP as an alternative substrate. The ratio of free HMC and cytosine released by MilB was ca. 9:1 in in vitro assays, and is consistent with the higher levels of MIL compared to dHM-MIL that are produced by Streptoverticillum rimofaciens.

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Section: Introductionsupporting

confidence: 67%

The Mildiomycin Biosynthesis: Initial Steps for Sequential Generation of 5‐Hydroxymethylcytidine 5′‐Monophosphate and 5‐Hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

et al. 2008

ChemBioChem

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“…Interestingly, although glucuronosyltransferases are commonly observed in mammalian metabolism, [21,22] few have been described in prokaryotic organisms. [23,24] To our knowledge, this is the first isolation of glucuronidated tetracycline biosynthetic intermediates, although glucosylated, truncated tetracycline intermediates have been reported to be produced by a S. rimosus mutant. [25] In summary, by using gene disruption and heterologous reconstitution experiments, we have characterized the function of OxyE as a C-4 monooxygenase during oxytetracycline biosynthesis in S. rimosus.…”

Section: Discussionmentioning

confidence: 93%

Identification of OxyE as an Ancillary Oxygenase during Tetracycline Biosynthesis

et al. 2009

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The double hydroxylation of 6-pretetramid to 4-keto-anhydrotetracycline is a key tailoring reaction during the biosynthesis of the broad-spectrum antibiotic tetracyclines. It has been shown previously by heterologous reconstitution that OxyL is a dioxygenase and is the only enzyme required to catalyze the insertion of oxygen atoms at the C-12a and C-4 positions. We report here that OxyE, a flavin adenine dinucleotide (FAD)-dependent hydroxylase homologue, is an ancillary mono-oxygenase for OxyL during oxytetracycline biosynthesis in Streptomyces rimosus. By using both gene disruption and heterologous reconstitution approaches, we demonstrated that OxyE plays a nonessential, but important role in oxytetracycline biosynthesis by serving as a more efficient C-4 hydroxylase. In addition, we demonstrated that partially oxidized biosynthetic intermediates can undergo various glycosylation modifications in S. rimosus. Our results indicate that the synergistic actions of OxyE and OxyL in the double hydroxylation step prevent accumulation of shunt products during oxytetracycline biosynthesis in S. rimosus.

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“…[10] Gould and co-workers were able to show that addition of exogenous cytosine to S. griseochromogenes culture media increased BS production by 1.6-fold and elevated the production of the intermediate cytosylglucuronic acid (CGA) by up to 70-fold. [11] These results were not surprising as intracellular concentrations of cytosine are likely quite low inasmuch as free cytosine is not an intermediate in pyrimidine biosynthesis. [12] The stimulation of BS production upon cytosine supplementation suggested that the availability of free cytosine might be a limiting factor in blasticidin biosynthesis.…”

Section: Introductionmentioning

confidence: 91%

Characterization of BlsM, a Nucleotide Hydrolase Involved in Cytosine Production for the Biosynthesis of Blasticidin S

Grochowski

Zabriskie

2006

ChemBioChem

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Biosynthesis of the antifungal agent blasticidin S in Streptomyces griseochromogenes requires the formation of free cytosine. The blsM gene in the blasticidin S gene cluster is predicted to encode a protein that has sequence homology with several nucleoside transferases. In vitro analysis of recombinant BlsM revealed that the enzyme functions as a nucleotide hydrolase and catalyzes the formation of free cytosine by using cytidine 5'-monophosphate (CMP) as the preferred substrate. Cytosine production was significantly lower with CDP, CTP, and dCMP as alternate substrates. BlsM was also observed to have low-level cytidine deaminase activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Point mutations were introduced in blsM at putative catalytic residues to generate three mutant enzymes, BlsM Ser98Asp, Glu104Ala, and Glu104Asp. All three mutants lost CMP hydrolysis activity, but the Ser98Asp mutant showed a modest increase in cytidine deaminase activity.

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Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase) from Streptomyces griseochromogenes, the first prokaryotic UDP-glucuronosyltransferase

Cited by 20 publications

References 33 publications

The Mildiomycin Biosynthesis: Initial Steps for Sequential Generation of 5‐Hydroxymethylcytidine 5′‐Monophosphate and 5‐Hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

The Mildiomycin Biosynthesis: Initial Steps for Sequential Generation of 5‐Hydroxymethylcytidine 5′‐Monophosphate and 5‐Hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

Identification of OxyE as an Ancillary Oxygenase during Tetracycline Biosynthesis

Characterization of BlsM, a Nucleotide Hydrolase Involved in Cytosine Production for the Biosynthesis of Blasticidin S

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