Biosynthesis of Streptolidine Involved Two Unexpected Intermediates Produced by a Dihydroxylase and a Cyclase through Unusual Mechanisms

Chang, Chin-Yuan; Lyu, S.Y.; Liu, Yu Chen; Hsu, N.S.; Wu, Chih Chung; Tang, Cheng Fong; Lin, Kuan Hung; Ho, Jin Yuan; Wu, Chang Jer; Tsai, Ming‐Daw; Li, Tsung Lin

doi:10.1002/anie.201307989

Cited by 54 publications

(53 citation statements)

References 33 publications

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“…An extract of the active site of OrfP is given in Figure 1 as taken from the 4M2E protein databank (PDB) file [11, 12] . The iron is linked to the protein through interactions with the side chains of His 154 , Glu 156 and His 303 groups in a typical facial 2‐His/1‐Glu triad, which is common for many nonheme iron dioxygenases [13] .…”

Section: Introductionmentioning

confidence: 99%

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

Ali

Henchman

Visser

2020

Chemistry A European J

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The nonheme iron enzyme OrfP reacts with l‐Arg selectively to form the 3R,4R‐dihydroxyarginine product, which in mammals can inhibit the nitric oxide synthase enzymes involved in blood pressure control. To understand the mechanisms of dioxygen activation of l‐Arg by OrfP and how it enables two sequential oxidation cycles on the same substrate, we performed a density functional theory study on a large active site cluster model. We show that substrate binding and positioning in the active site guides a highly selective reaction through C3−H hydrogen atom abstraction. This happens despite the fact that the C3−H and C4−H bond strengths of l‐Arg are very similar. Electronic differences in the two hydrogen atom abstraction pathways drive the reaction with an initial C3−H activation to a low‐energy 5σ‐pathway, while substrate positioning destabilizes the C4−H abstraction and sends it over the higher‐lying 5π‐pathway. We show that substrate and monohydroxylated products are strongly bound in the substrate binding pocket and hence product release is difficult and consequently its lifetime will be long enough to trigger a second oxygenation cycle.

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Section: Introductionmentioning

confidence: 99%

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

Ali

Henchman

Visser

2020

Chemistry A European J

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“…Interestingly, an arginine guanidino moiety corresponding to the lysine -amino group was surrounded by two acidic residues (D268 and D270 in VioC), suggesting that one or both acidic residues act as substrate acceptors. Subsequent mutagenesis experiments demonstrated that both residues were likely important for activity, but K3H-1 D241 and K4H-4 D253, rather than D243 and E256, respectively, appear to be more critical for the interaction with L-lysine ( 27), daptomycin-type antibiotic (AsnO) (9), and streptolidine (OrfP) (28). On the other hand, L-isoleucine hydroxylase functions as a tricarboxylic acid cycle shunt and in 2-amino-3-methyl-4-ketopentanoic acid biosynthesis, which serves as a metabolic antagonist during vitamin B 12 production (29).…”

Section: Resultsmentioning

confidence: 99%

Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction

Hara

Yamagata

Miyake

et al. 2017

Appl Environ Microbiol

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Hydroxylation via C-H bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available l-lysine with l-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two l-lysine 3-hydroxylases and four l-lysine 4-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereoselective hydroxylation into either C-3 or C-4 positions of l-lysine, yielding (2,3)-3-hydroxylysine and (2,4)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant l-lysine hydroxylase-expressing cells were used as a biocatalyst for l-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2,3)-3-hydroxylysine was produced from 600 mM l-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2,4)-4-hydroxylysine was produced from 300 mM l-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies. The present study identified six l-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of l-lysine hydroxylases was relatively narrow, we found that (2,3)-3-hydroxylysine was hydroxylated by 4-hydroxylase and (2,5)-5-hydroxylysine was hydroxylated by both 3- and 4-hydroxylases. Moreover, the l-arginine hydroxylase VioC also hydroxylated l-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of l-lysine into (2,3)-3-hydroxylysine and (2,4)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic l-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

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“…Recently, two research groups reported on genes responsible for biosynthesis of the streptolidine lactam and amino sugar moieties in ST (26,27). The orfP and orfR genes from the ST producer Streptomyces lavendulae BCRC 12163 were found to participate in the early steps of the streptolidine lactam biosynthesis (26).…”

Section: Identification Of the Bd-12-biosynthetic Gene Cluster From Smentioning

confidence: 99%

“…The orfP and orfR genes from the ST producer Streptomyces lavendulae BCRC 12163 were found to participate in the early steps of the streptolidine lactam biosynthesis (26). The stnG, stnI, stnJ, and stnQ genes from the ST producer Streptomyces sp.…”

Section: Identification Of the Bd-12-biosynthetic Gene Cluster From Smentioning

confidence: 99%

tRNA-Dependent Aminoacylation of an Amino Sugar Intermediate in the Biosynthesis of a Streptothricin-Related Antibiotic

Maruyama

Niikura

Izumikawa

et al. 2016

Appl Environ Microbiol

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The antibiotic streptothricin (ST) possesses an amino sugar bound to an L-␤-lysine (␤-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a ␤-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics. (1), STs with an oligo(␤-Lys) consisting of two to seven residues have been identified (Fig. 1). ST-F inhibits protein biosynthesis in prokaryotic cells (2), and STs carrying the longer oligo(␤-Lys) side chains show higher levels of antibacterial activity. Moreover, STs strongly inhibit the growth of eukaryotes, such as yeasts (3-5), fungi (6), protozoa (7), insects (8), plants (9), and mammals (10-13). Although STs have been used effectively as selective agents for recombinant DNA work in some of these organisms, STs are not currently used therapeutically due to their inherent toxicity. In addition to the STs, it has been reported that Streptomyces strains produce ST analogues that possess a glycinederived side chain rather than the ␤-Lys residue: 15), citromycin (16,17), glycinothricin (18), A-269A (19), and A-269A= (19) (Fig. 1). These analogues display potent antibacterial activities, although their molecular targets remain unclear. Also, like the STs, the ST analogues are not used clinically due to their toxicity.We previously identified the ST-biosynthetic gene cluster (accession no. AB684619) in Streptomyces rochei NBRC 12908 (Fig. 2) and elucidated the biosynthetic mechanisms of the oligo(␤-Lys) side chains (20). Nonribosomal peptide synthetases (NRPSs) are known to catalyze the assembly of a myriad of structurally complex peptide natural products (21). However, in the previous study, we identified three unique stand-alone NRPSs among the ST-biosynthetic enzymes and showed that they assembled the structurally simple peptide oligo(␤-Lys) (Fig. 3A). The biosynthesis is initiated by adenylation of ␤-Lys in Orf5 (stand-alone adenylation [A] domain), and the resulting L-␤-lysyl-O-AMP is loaded

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Biosynthesis of Streptolidine Involved Two Unexpected Intermediates Produced by a Dihydroxylase and a Cyclase through Unusual Mechanisms

Cited by 54 publications

References 33 publications

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction

tRNA-Dependent Aminoacylation of an Amino Sugar Intermediate in the Biosynthesis of a Streptothricin-Related Antibiotic

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