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Discovery of an unprecedented hydrazine-forming machinery in bacteria Kenichi Matsuda, Takeo Tomita, Kazuo Shin-ya, Toshiyuki Wakimoto, Tomohisa Kuzuyama, and Makoto Nishiyama J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05354 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018
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Journal of the American Chemical Society
Discovery of an unprecedented hydrazine-forming machinery in bacteria Kenichi Matsuda1,2, Takeo Tomita1,3, Kazuo Shin-ya1,4, Toshiyuki Wakimoto2, Tomohisa Kuzuyama1,3, Makoto Nishiyama*1,3 1
Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan 3 Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo 113-8657, Japan 4 National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan 2
Supporting Information Placeholder ABSTRACT: Recent studies described several different routes that facilitate nitrogen-nitrogen bond formation in natural product biosynthesis. We herein report the identification of an unprecedented machinery for hydrazine formation involved in the biosynthesis of s56-p1, a dipeptide natural product with a unique hydrazone unit. The gene cassette comprising this machinery is widespread across several bacterial phyla, highlighting overlooked potential of bacteria to synthesize hydrazine.
disrupted each gene from spb37 to spb50 and examined N2H4 generation with acid hydrolysis. The results obtained showed that the disruption of two genes, spb38 encoding a putative Nhydroxylase and spb40 coding for a fusion protein consisting of a cupin and methionyl-tRNA synthetase (metRS)-like protein, caused the complete loss of N2H4 generation (Supplementary Fig. 1c). a)
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Natural products containing a nitrogen-nitrogen (N-N) bond are structurally diverse and exhibit many biological activities1,2. An increasing number of biosynthetic gene clusters of natural products containing diazo3-9, azoxy10-18, hydrazide19-29, hydrazine30-33, hydrazone34 and pyridazine35,36, have been identified from actinomycetes. Studies on these gene clusters show that nature exploits several different strategies to form N-N bonds37; however, the mechanisms responsible for N-N bond formation have not yet been elucidated in detail in most cases. s56-p1, a dipeptide compound with a unique hydrazone unit, was recently discovered from Streptomyces sp. SoC090715LN17, using a genome mining approach targeting amino-group carrier protein genes34. The s56-p1 biosynthetic gene cluster is the first gene cluster identified to be responsible for the biosynthesis of hydrazone-containing natural products (Figure 1a). Hereafter, we refer this as spb (s56-p1 biosynthesis) gene cluster. We previously performed gene knockout experiments and revealed that a genetic region composed of eight open reading frames (spb37-spb44, described as orf37-orf44 in previous report) was involved in the biosynthesis of the hydrazone unit of s56-p1. We herein report the characterization of Spb38, Spb39, and Spb40. The combination of in vivo and in vitro experiments showed that these enzymes cooperate to synthesize hydrazinoacetic acid (HAA), a putative precursor for the hydrazone unit of s56-p1. Notably, the subset of genes comprising the HAA synthetic machinery is widely distributed across taxonomically distinct bacterial species. We introduced a partial gene cluster containing spb37-spb50 into the heterologous host Streptomyces lividans TK23, and detected N2H4 in the acid hydrolysate of the culture broth of the transformant (Supplementary Fig. 1a, b), which is consistent with the findings of a previous gene deletion experiment34. In order to identify the key enzyme involved in N-N bond formation, we
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Figure 1. (a) spb cluster, with spb38, spb39, and spb40 highlighted. (b) LC-MS analysis of E. coli expressing spb38 and spb40. pRSF is a duet vector for co-expression of two genes in one host organism. (c) Chemical structure of 1-DNP. Spb38 is homologous to lysine N-hydroxylases, such as LucD, PvdA, and ornithine N-hydroxylase KtzI. LucD and PvdA are involved in siderophore biosynthesis in Escherichia coli38 and Pseudomonas aeruginosa39, respectively, while KtzI is involved in the biosynthesis of piperazate (Piz), a building block with the N-N bond for cyclic peptide kutzneride24. They all catalyzed the hydroxylation of side-chain primary amines in FAD- and oxygendependent manners using NAD(P)H as the electron donor. In order to characterize the putative monooxygenase Spb38 experimentally, an in vitro reaction with recombinant Spb38 was performed under aerobic conditions using L-lysine as a substrate. The reaction product of NbtG: N6- L-lysine monooxygenase in Nocobactin biosynthesis from Nocardia farcinica40 was used as the standard. In order to readily detect the product, the reaction mixture was derivatized by 9-fluorenylmethyl chloroformate (Fmoc) prior to detection. A HPLC analysis revealed the efficient consumption of L-lysine and generation of N6-OH L-lysine in the presence of FAD and NADPH (Supplementary Fig. 2). Spb38 also utilized NADH as a reducing equivalent. These results indi-
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cate that Spb38 catalyzes the hydroxylation of N6 of L-lysine in FAD- and NAD(P)H-dependent manners. We then investigated the function of the protein encoded in spb40. Spb40 is a di-domain protein consisting of the N-terminal cupin domain and C-terminal metRS-like domain. In order to obtain functional insights, spb40 was expressed in E. coli together with spb38. An LC-MS analysis of the metabolites of recombinant E. coli cells revealed the accumulation of compound 1 (m/z 220.1286) with the molecular formula C8H18N3O4 (calcd. as m/z 220.1292 [M+H]+) (Figure 1b), which was not observed in the culture broth of E. coli cells lacking spb38 or spb40. In order to incorporate 15N atoms into 1, production media were supplemented with 15NH4Cl. An LC-MS analysis of the culture broth showed that a maximum of three 15N atoms were incorporated into 1 (Supplementary Fig. 3). Attempts to isolate 1 were hampered by its high hydrophilicity and instability under acidic conditions. In order to overcome this, we conducted the derivatization of 1 with 2,4-dinitrofluorobenzene (DNFB) to increase its hydrophobicity and isolated the 1-dinitrophenyl (DNP) conjugate instead of intact 1. 15N-labeled 1-DNP conjugates were isolated by anion exchange and reverse phase column chromatography, and then subjected to NMR analyses (Supplementary Figs. 4-8). The interpretation of 1 H and 13C data allowed the assignment of two separate spin systems as N-DNP-L-lysine and N-DNP-glycine. 1H-15N HMBC data clearly showed interresidual cross-peaks between L-lysine and glycine (Supplementary Fig. 8). Therefore, L-lysine and glycine units were connected via hydrazine linkage, and the unprecedented structure of 1-DNP was determined (Figure 1c). A feeding study with deuterium-labeled L-lysine, glycine, or both resulted in the incorporation of deuterium atoms into 1, unambiguously showing that 1 is derived from these two amino acids (Supplementary Fig. 9). The production of 1 was detected when the culture broth of E. coli cells expressing spb40 was supplemented with the in vitro reaction mixture of spb38 containing N6-OH Llysine (Supplementary Fig. 10). Therefore, L-lysine was initially hydroxylated by the function of Spb38, and the resultant N6-OH L-lysine was then conjugated with glycine via a hydrazine linkage to form 1 by the function of Spb40. The formation of N-N bond by Spb40 may proceed via either of two hypothetical mechanisms as follows: one is the intermolecular N-N bond-forming pathway in which the hydroxylamine group at N6 of L-lysine is activated by phosphorylation or adenylation to facilitate the nucleophilic attack from the amino group of glycine (Figure 2a). The other mechanism is the N-N bond-forming pathway proceeding via the formation of an ester intermediate generated by the conjugation of N6-OH L-lysine and glycine (Figure 2b). In order to identify which pathway is employed to synthesize 1, we conducted a feeding experiment of N6-18OH L-lysine. This was prepared in vitro by the enzymatic reaction of Spb38 in the presence of 18O2 (Supplementary Fig. 11), and the reaction mixture was added to the culture broth of E. coli expressing spb40 for bioconversion. An LC-MS analysis of the resultant culture broth detected the incorporation of an 18O atom into 1 (Figure 2c). Furthermore, an MS/MS analysis showed that the 18O was incorporated into the carboxy group of the glycine molecule of 1 (Figure 2d, e). This result strongly suggested that Spb40 synthesized 1 via the pathway described in Figure 2b, in which the N-N bond was formed via the rearrangement of the putative ester intermediate. The C-terminal domain of Spb40 (Spb40-C) exhibited strong similarity to metRS, which catalyzes the adenylation of methionine and its loading on its cognate tRNA. The motif for ATP binding41 was also present in Spb40-C (Supplementary Fig. 12a), which may be involved in the activation of the glycine carboxyl group. When this motif was mutated by alanine substitution, E. coli expressing this variant abolished the production of 1 (Supplementary Fig. 13). It currently remains unclear whether Spb40-
C requires tRNA as a substrate because in contrast to metRS, three amino acid residues that are tightly conserved in the anticodon-binding domain for the recognition of tRNAMet42 were absent in Spb40-C (Supplementary Fig. 12a), suggesting its unique function and distinct substrate specificity from those of canonical metRSs. Although the formation of the ester intermediate followed by its rearrangement remains elusive, the N-terminal cupin domain (Spb40-N) may be responsible for this transformation. Cupin superfamily proteins catalyze a wide range of reactions by exploiting a metal center. However, cupin has not yet been demonstrated to catalyze N-N bond formation. Multiple sequence alignments showed that the metal-binding site43 consisting of four amino acid residues (Asp43, His45, Glu49, His83) is present in Spb40-N (Supplementary Fig. 12b). E. coli producing the Spb40 mutant with the substitution of alanine for His45 abolished the ability to produce 1 (Supplementary Fig. 13). Thus, these sites should be involved in formation and successive rearrangement of the ester intermediate suggested by stable isotope experiments.
Figure 2. Models for the biosynthesis of 1 from N6-OH L-lysine and glycine and feeding studies on N6-18OH L-lysine. Models for the biosynthesis of 1 (a) via intermolecular N-N bond formation through the activation of hydroxylamine and (b) via the formation of the putative ester intermediate followed by its rearrangement. The labeled oxygen in these models is highlighted in red. (c) An LC-MS analysis of the culture broth of E. coli::pRSF40 supplemented with none (black, bottom), with the Spb38 in vitro reaction mixture containing N6-OH L-lysine (black, middle), and with that containing N6-18OH L-lysine (red), is described. (d) MS/MS spectra of 1 (black) and 18O-labeled 1 (red). (e) Assignment of fragments in the structure of 1. A fragment labeled with a stable isotope was highlighted. Spb39 is homologous to D-amino acid oxidase (DAO), which catalyzes the oxidation of Cα-N bonds to yield an imino group in an FAD-dependent manner. Oxidation is commonly followed by hydrolysis of the imino acid to afford α-keto acid and amine. The spb39 gene often co-localizes with spb38 and spb40 genes on the genomes of various bacteria, which prompted us to postulate that Spb39 may catalyze a reaction using 1 as a substrate. Similar to
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Journal of the American Chemical Society DAO family proteins, purified Spb39 contained FAD (Supplementary Fig. 14). Recombinant Spb39 was added to the filtered culture broth of E. coli producing 1, together with 0.1 mM FAD, and incubated, and the reaction product was derivatized with DNFB. A successive LC-MS analysis showed the consumption of 1 with m/z 522.1321 and the generation of a new compound with a molecular ion peak at m/z 257.0513, corresponding to the molecular formula of C8H9N4O6 (calcd. as m/z 257.0517 [M+H]+) (Figure 3a, b). The product was identified as hydrazinoacetic acid (HAA) modified with DNFB (HAA-DNP) by an LC-MS/MS analysis with the authentic sample (Figure 3b). The timedependent consumption of 1 and generation of HAA was observed by a HPLC analysis. (Supplementary Fig. 15). When the reaction mixture of Spb39 was treated with NaBH4, we observed pipecolate, which may accumulate by the reduction of ∆1piperadine-6-carboxylic acid: P6C (Supplementary Fig. 16). P6C is a stable compound that is spontaneously generated by the cyclization of 2-aminoadipate 6-semialdehyde (AASA). In addition, an in vitro experiment using [3,3,4,4,5,5,6,6,9,9-D10] 1 resulted in the disappearance of one deuterium atom in the pipecolate of the NaBH4-treated reaction mixture with two deuterium atoms retained in HAA, which indicated the oxidation of the C6-N bond by Spb39 (Supplementary Fig. 17). Collectively, these results indicate that Spb39 oxidizes 1 to yield HAA and AASA in an FAD-dependent manner. a) EICs of m/z 552.1321
enzymes involved are different between HAA and valanimycin biosynthesis, further studies on Spb40 may provide insight into the logic of N-N bond formation conserved in biosynthesis of other natural products. The HAA biosynthetic pathway shown in Figure 3c was for the first time characterized for spb cluster, suggesting the uniqueness of this machinery in natural product biosynthesis. However, a BLAST search revealed that similar gene cassettes are widely distributed not only among Actinobacteria, but also among various bacterial species from several different phyla: Proteobacteria, Firmicutes, Deinococcus-Thermus, and Cyanobacteria (Supplementary Fig. 18a). This result is in contrast to actinomycetes being a major bacterial source for N-N bond-containing natural products. In some cases, the gene cassettes for HAA biosynthesis co-localize with genes that are presumably involved in secondary metabolite biosynthesis such as non-ribosomal peptide synthetases, suggesting that HAA is also utilized as a common intermediate of unidentified natural products in the strains (Supplementary Fig. 18b). Therefore, this study revealed an overlooked potential of bacteria to synthesize hydrazine compounds and will facilitate a genome-mining approach to discover new natural products derived from HAA.
ASSOCIATED CONTENT
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Supporting Information 1-DNP (std.)
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The Supporting Information is available free of charge on the ACS Publications website. The strains, plasmids, and oligonucleotides used in the present study were listed in Tables S1, S2, and S3, respectively, in supporting information. Detailed materials and methods are provided in supporting information.
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AUTHOR INFORMATION Corresponding Author
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Figure 3. (a), (b) LC-MS analysis of the in vitro Spb39 reaction mixture. (c) Biosynthetic pathway of HAA from L-lysine and glycine mediated by Spb38, Spb39, and Spb40. Although the enzymes responsible for N-N bond formation have not yet been understood in detail, nature appears to exploit several different strategies, either enzymatic or non-enzymatic, to form N-N bonds in natural product biosynthesis. KtzT was very recently demonstrated in vitro to catalyze hydrazine-bond formation in the biosynthesis of Piz25. Similar to HAA biosynthesis, Piz biosynthesis starts from the hydroxylation of the primary amine by KtzI, which is homologous to Spb38. However, the subsequent chemical steps differ between the two pathways. In the Piz pathway, the iron center of the heme-dependent enzyme KtzT appears to polarize the substrate N-O bond of N5-OH ornithine to facilitate an intramolecular cyclization to form N-N bond. On the other hand, HAA biosynthesis may employ alternative mechanisms that are in part similar to valanimycin biosynthesis where N-N bond formation starts from the hydroxylation of the nitrogen atom of isobutylhydroxylamine, followed by transfer of a serine residue from seryl-tRNA to form the ester intermediate13. However, following further oxidation and intramolecular rearrangement steps to form N-N bond remain unclear. Although the
The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported in part by JSPS KAKENHI Grant Nos. 24228001 and 17H06168 (M. Nishiyama) and the Japan Foundation for Applied Enzymology (M. Nishiyama). This study was also funded by the MEXT-supported Program for Strategic Research Foundation at Private Universities, 2013–2017 (S1311017), as well as a grant for “Project focused on developing key technology of discovering and manufacturing drug for nextgeneration treatment and diagnosis” from METI, Japan (to K. Shin-ya and T. Kuzuyama). We thank Professor Haruo Ikeda (Kitasato University) for providing us with the λ Red recombination system, pKU487, pRED and pKU1021.
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