C3′‐Deoxygenation of Paromamine Catalyzed by a Radical <i>S</i>‐Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3

Kim, Hak Joong; LeVieux, Jake A.; Yeh, Yu-Cheng; Liu, Hung‐wen

doi:10.1002/ange.201510635

Cited by 8 publications

(14 citation statements)

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“…Several dehydration mechanisms from the C4′-radical intermediate have been proposed by Liu and coworkers. 6 Any reaction mechanisms must introduce one hydrogen atom into the C3′ moiety of 4′-oxolividamine. When we conducted the AprD4/AprD3 reaction with 1 in the presence of D 2 O, a deuterium atom was incorporated into the equatorial position at C3′ of 7 (Supplementary Figure S6).…”

Section: Resultsmentioning

confidence: 99%

“…12 Liu and coworkers showed that the H4′ atom of 1 is abstracted by 5′-dAdo• to give 5′-deoxyadenosine (5′-dAdo) as one of products in addition to the dehydrated product (Figure 2). 6 The reaction of AprD4/AprD3 with 1, 2, kanamycin C (3) and kanamycin B (4) produced similar amounts of 5′-dAdo, whereas only a small amount of 5′-dAdo was detected with 2′-N-acetylparomamine (5) and paromomycin (6) (Figure 3 and Table 1). Without substrate, only trace amount of 5′-dAdo formation was detected (data not shown).…”

Section: Enzyme Reaction In the Presence Of Deuterium Oxidementioning

confidence: 99%

“…Presumably, a putative common biosynthetic intermediate lividamine (3′-deoxyparomamine) is involved in both tobramycin and apramycin biosynthetic pathways. Liu and coworkers 6 have recently shown that both AprD4 and AprD3 are responsible for the C3′-deoxygenation of 1 to give lividamine (7) using in vitro enzymatic analysis (Figure 2). They observed that the H4′ of 1 is abstracted by a 5′-deoxyadenosyl radical generated from SAM to initiate this unique radical-mediated dehydration reaction (Supplementary Figure S1).…”

Section: Introductionmentioning

confidence: 99%

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Substrate specificity of radical S-adenosyl-l-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics

2016

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A radical S-adenosyl-L-methionine dehydratase AprD4 and an NADPH-dependent reductase AprD3 are responsible for the C3′-deoxygenation of pseudodisaccharide paromamine in the biosynthesis of apramycin. These enzymes are involved in the construction of the characteristic structural motif that is not modified by 3′-phosphotransferase in aminoglycoside-resistant bacterial strains. AprD4 catalyzes the C3′-dehydration of paromamine via a radical-mediated reaction mechanism to give 4′-oxolividamine, which is then reduced by AprD3 with NADPH to afford lividamine. In the present study, the substrate specificity of this unique combination of enzymes has been investigated. AprD4 was found to recognize paromamine, neamine, kanamycin C, and kanamycin B to afford 5′-deoxyadenosine as one of products during the C3′-dehydration of aminoglycosides, but not 2′-N-acetylparomamine and paromomycin. Only paromamine and kanamycin C were converted to the corresponding C3′-deoxygenated compounds by AprD4 and AprD3. AprD3 recognizes the 4′-oxolividamine moiety, including the pseudotrisaccharide kanamycin C, and seems to reject the amino group at C6′ of neamine and kanamycin B. Chirally deuterium-labeled NADPH was used to identify that that AprD3 transfers the pro-S hydrogen atom of NADPH when reducing 4′-oxolividamine to give lividamine.

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Section: Resultsmentioning

confidence: 99%

Section: Enzyme Reaction In the Presence Of Deuterium Oxidementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Substrate specificity of radical S-adenosyl-l-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics

2016

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“…Interestingly, structurally related C-3' deoxygenation and C-3',4'-dideoxygenation of AGAs are catalyzed by distinct catalytic pathways. C-3' deoxygenation is catalyzed through a radical mechanism by the radical SAM dehydratase, AprD4, along with the reductase partner, AprD3 [12][13][14][15] . Although an AprD3 homologue was identified in the gentamicin pathway, GenB3-catalyzed dehydration and 3'-phosphate elimination do not require a reductase partner.…”

Section: Discussionmentioning

confidence: 99%

“…The C-3' deoxygenation biosynthetic process has recently been dissected by several research groups [12][13][14] . A radical S-adenosyl-L-methionine (SAM) enzyme AprD4, along with AprD3, is responsible for the 3'-deoxygenation in apramycin biosynthesis 15 .…”

Section: Introductionmentioning

confidence: 99%

Pyridoxal-5'-Phosphate-Dependent Enzyme GenB3 Catalyzes C-3',4'-Dideoxygenation in Gentamicin Biosynthesis

Zhou

Chen

et al. 2020

Preprint

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Background: C-3',4'-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3'-phosphotransferase (APH(3')) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. Results: Here, we report GenP catalyzes 3′ phosphorylation of gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that a pyridoxal-5′-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3',4'-dideoxy-4',5'-ene-6'-oxo products. The following C-6' transamination and GenB4 catalyzed reduction of 4',5' olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. Conclusions: This discovery solves the long-standing puzzle in gentamicin biosynthesis, also enriches the chemistry of PLP dependent enzymes. Interestingly, these results demonstrate that to evade APH(3') deactivation from the pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3') to activate hydroxyls as leaving groups for the 3',4'-dideoxygenation in gentamicin biosynthesis.

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Two Cryptic Self‐Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin

Zhang

Chi

et al. 2021

Angewandte Chemie

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Apramycin is aclinically promising aminoglycoside antibiotic (AGA). To date,m echanisms underlying the biosynthesis and self-resistance of apramycin remain largely unknown. Here we report that apramycin biosynthesis proceeds through unexpected phosphorylation, deacetylation, and dephosphorylation steps,i nw hich an ovel aminoglycoside phosphotransferase (AprU), ap utative creatinine amidohydrolase (AprP), and an alkaline phosphatase (AprZ) are involved. Biochemical characterization revealed that AprU specifically phosphorylates 5-OH of ap seudotrisaccharide intermediate,w hose N-7' acetyl group is subsequently hydrolyzed by AprP.A prZ is located extracellularly where it removes the phosphate group from ap seudotetrasaccharide intermediate,leading to the maturation of apramycin. Intriguingly,7'-N-acetylated and 5-O-phosphorylated apramycin that were accumulated in DaprU and DaprZ respectively exhibited significantly reduced antibacterial activities,i mplying Streptomyces tenebrarius employs C-5 phosphorylation and N-7' acetylation as two strategies to avoid auto-toxicity.S ignificantly,t his study provides insight into the design of new generation AGAs to circumvent the emergence of drugresistant pathogens.

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C3′‐Deoxygenation of Paromamine Catalyzed by a Radical S‐Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3

Cited by 8 publications

References 50 publications

Substrate specificity of radical S-adenosyl-l-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics

Substrate specificity of radical S-adenosyl-l-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics

Pyridoxal-5'-Phosphate-Dependent Enzyme GenB3 Catalyzes C-3',4'-Dideoxygenation in Gentamicin Biosynthesis

Two Cryptic Self‐Resistance Mechanisms in Streptomyces tenebrarius Reveal Insights into the Biosynthesis of Apramycin

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