Pyridoxal-5′-phosphate as an oxygenase cofactor: Discovery of a carboxamide-forming, α-amino acid monooxygenase-decarboxylase

Ying Huanga,b, Xiaodong Liua, Zheng Cuia, Daniel Wiegmannc, Giuliana Niroc, Christian Duchoc, Yuan Songb, Zhaoyong Yangd,1, and Steven G. Van Lanena,1

aDepartment of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536; bDepartment of Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100094, People’s Republic of China; cDepartment of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, 66123 Saarbrücken, Germany; and dInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved December 18, 2017 (received for review October 25, 2017) Capuramycins are antimycobacterial antibiotics that consist of a The A-500359s from Streptomyces griseus SANK 60196 (5), the A- modified nucleoside named uridine-5′-carboxamide (CarU). Previ- 503083s from Streptomyces sp. SANK 62799 (6), and the A-102395 ous biochemical studies have revealed that CarU is derived from from Amycolotopsis sp. SANK 60206 (7) are capuramycin-type UMP, which is first converted to uridine-5′-aldehyde in a reaction secondary metabolites (8) that are potent inhibitors of bacterial catalyzed by the dioxygenase CapA and subsequently to 5′-C- translocase I and have promising antimycobacterial activity. This glycyluridine (GlyU), an unusual β–hydroxy-α-amino acid, in a re- group of nucleoside antibiotics is characterized by two rarely ob- action catalyzed by the pyridoxal-5′-phosphate (PLP)-dependent served structural components: a uridine-5′-carboxamide (CarU) transaldolase CapH. The remaining steps that are necessary to nucleoside core and an unsaturated hexuronic acid appended to furnish CarU include decarboxylation, O atom insertion, and oxi- the core through a 5′-O-glycosidic bond (Fig. 1). The hexuronic acid dation. We demonstrate that Cap15, which has sequence similarity is subsequently modified with a third component, an L-amino- to proteins annotated as bacterial, PLP-dependent L-seryl-tRNA caprolactam in the case of A-500359s/A-503083s and an unusual (Sec) selenium transferases, is the sole catalyst responsible for arylamine-containing polyamide in the case of A-102395, both of complete conversion of GlyU to CarU. Using a complementary which are attached to the hexuronic acid via an amide bond (Fig. 1). panel of in vitro assays, Cap15 is shown to be dependent upon The biosynthetic gene clusters for all three capuramycin-type me- substrates O and (5′S,6′R)-GlyU, the latter of which was unex- 2 tabolites have been cloned and characterized (9–11). Comparative pected given that (5′S,6′S)-GlyU is the isomeric product of the transaldolase CapH. The two products of Cap15 are identified as bioinformatic analysis revealed 18 homologous open reading frames (orfs) between the capuramycin clusters, consistent with an impor- the carboxamide-containing CarU and CO2. While known enzymes that catalyze this type of chemistry, namely α-amino acid 2-mono- tant role in resistance, regulation, and the biosynthesis of the shared oxygenase, utilize flavin adenine dinucleotide as the redox cofac- CarU-hexuronic acid disaccharide core (11). tor, Cap15 remarkably requires only PLP. Furthermore, Cap15 does Of the 18 shared orfs in the gene clusters for the capuramycin- not produce hydrogen peroxide and is shown to directly incorpo- type metabolites, two encode sequence-unrelated proteins that are each predicted to be PLP-dependent enzymes: CapH, with rate a single O atom from O2 into the product CarU and thus is an authentic PLP-dependent monooxygenase. In addition to these un- usual discoveries, Cap15 activity is revealed to be dependent upon Significance the inclusion of phosphate. The biochemical characteristics along with initiatory mechanistic studies of Cap15 are reported, which Enzymes that activate dioxygen typically rely on flavins or has allowed us to assign Cap15 as a PLP-dependent (5′S,6′R)-GlyU: transition metals as redox cofactors. We describe the discovery O2 monooxygenase-decarboxylase. of the enzyme catalyst Cap15 that converts 5′-C-glycyluridine (GlyU), an unusual β–hydroxy-α-amino acid, to the modified natural products | biosynthesis | antibiotic | enzyme function nucleoside uridine-5′-carboxamide. In contrast to expectations, pyridoxal-5’-phosphate (PLP) is the sole cofactor that serves as yridoxal-5′-phosphate (PLP)-dependent enzymes account for the initiating reducing agent to activate dioxygen for in- Pmore than 230 distinct catalytic functions, most of which in- corporation into GlyU prior to decarboxylation. Thus, Cap15 is volve transamination, decarboxylation, deamination, or racemiza- now classified as an O2- and PLP-dependent monooxygenase- tion of α-amino acids (1). Although ostensibly less common, other decarboxylase. Also in contrast to expectations, phosphate and types of PLP-dependent enzymes are known, including those cat- the (5′S,6′R)isomerofGlyU—and not (5′S,6′S)-GlyU, the alyzing β-orγ-elimination or -substitution, aldol- and Claisen-type established pathway intermediate—are essential for Cap15 activity. In total, Cap15 utilizes an enzymatic strat- reactions, and even reactions involving O2-dependent oxidation (2– 4). Collectively, PLP-dependent enzymes currently occupy five of egy for oxygen incorporation during conversion of an α the six classes used by the Enzyme Commission, with synthetases -amino acid to a carboxamide. the lone category yet to be reported. The power and versatility — Author contributions: Y.H., X.L., D.W., G.N., C.D., Y.S., Z.Y., and S.G.V.L. designed re- inherent in PLP is chiefly due to the ability to form an imine first search; Y.H., X.L., Z.C., D.W., G.N., and S.G.V.L. performed research; Y.H., X.L., C.D., and with the side-chain amine of a conserved Lys of the enzyme to form S.G.V.L. analyzed data; and Y.H., X.L., G.N., C.D., and S.G.V.L. wrote the paper. aninternalaldimineandthenwithasubstratetoformanexternal The authors declare no conflict of interest. — aldimine and subsequently serve as an electron sink during the This article is a PNAS Direct Submission. chemical transformation of the substrate. Despite steady advances Published under the PNAS license. in our understanding of the factors dictating the chemistry down- 1To whom correspondence may be addressed. Email: [email protected] or svanlanen@ stream of aldimine formation (2), the prediction of a PLP- uky.edu. — dependent enzyme activity solely based on sequence remains for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the most part—an inexact science. 1073/pnas.1718667115/-/DCSupplemental.

974–979 | PNAS | January 30, 2018 | vol. 115 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1718667115 Downloaded by guest on September 23, 2021 catalyzed transformation, adding to the already remarkable catalytic diversity of PLP-dependent enzymes found in living systems. Results Bioinformatic Analysis of Cap15. The gene product of cap15 has moderate sequence identity (45–50%) to proteins annotated as putative L-seryl-tRNA(Sec) selenium transferase, SelA. Sec- ondary structure-based predictions mirrored this analysis with the highest similarity to Aquifex aeolicus SelA (13), although sequence identity by pairwise alignment was significantly lower (24%, SI Appendix, Fig. S1A). Bacterial SelA catalyzes the PLP- dependent production of selenocysteinyl-tRNA(Sec) and phos- phate from substrates seryl-tRNA(Sec) and selenophosphate (SI Appendix, Fig. S2). Studies with AaSelA have revealed that this enzyme functions as a pentamer of dimers, with residues from each dimer subunit (termed A and B subunits) contributing to catalysis (13), and the reaction is initiated by formation of an internal aldimine between Lys285A and PLP. Based on sequence comparison, Lys230 is predicted to be the respective residue in Cap15 (SI Appendix, Fig. S1A). Only two additional Lys residues are conserved (Lys318 and Lys362 of AaSelA corresponding to Lys265 and Lys303 of Cap15, respectively). As part of the B sub- unit, the former Lys is located within ∼12 Å of the PLP and selenophosphate binding site of the A subunit; in contrast, the latter Lys is located distant from the active site. The importance of either residue has not been established. The B subunit of the Fig. 1. Structure and biosynthesis of capuramycin-type metabolites. Pro- AaSelA dimer also has two essential Arg residues that are pro- posed biosynthesis of the shared CarU core of capuramycin-type metabolites A-500359s and A-503083s starting from UMP. CapA and CapH, along with posed to establish an important electrostatic interaction for the homologs LipL and LipK from the biosynthetic pathway for A-90289 that binding of PLP and the substrate selenophosphate (SI Appendix, contains an intact (5′S,6′S)-GlyU in the final metabolite, have been previously Fig. S1B). While one of the Arg residues is conserved in Cap15 259 262 assigned in vitro. *LipK was previously shown to stereospecifically generate (Arg ), the second is substituted with a Lys (Lys )(SI Ap- (5′S,6′S)-GlyU. αKG, α-ketoglutarate. pendix, Fig. S1A).

Functional Assignment of Cap15. Recombinant His6-Cap15 was primary sequence similarity to proteins annotated as serine soluble upon production in Escherichia coli and purified to near hydroxymethyltransferases, and ORF15, with sequence similarity homogeneity using immobilized metal affinity chromatography to bacterial proteins annotated as L-seryl-tRNA(Sec) selenium (SI Appendix, Fig. S3A). Cap15(K230A) was also prepared as a transferases, SelA. CapH—the discovery of which added to the potential control (SI Appendix, Fig. S3A). Both purified protein catalog of reported PLP-dependent catalytic activities—was solutions were colorless and lacked a UV/Vis spectrum in- assigned as a transaldolase that generates acetaldehyde and 5′-C- dicative of a PLP cofactor. The hypothetical substrate (5′S,6′S)- glycyluridine (GlyU) from L-Thr and uridine-5′-aldehyde (U5′ GlyU, which was identified as the stereoisomer produced by the A), the last of which is generated from UMP in a reaction cat- CapH-homolog LipK (47% sequence identity via pairwise alyzed by a nonheme Fe(II)-dependent α-ketoglutarate:UMP alignment) that is involved in the biosynthesis of A-90289 (Fig. 1) dioxygenase CapA (Fig. 1) (11, 12). The uncovering of the tan- (14), was synthesized (SI Appendix, SI Materials and Methods dem reactions catalyzed by CapA and CapH revealed GlyU to be and Fig. S4) and tested with Cap15 in Hepes buffer with PLP. the likely biosynthetic precursor of CarU, which was further Following overnight reactions, HPLC analysis did not reveal any supported by isotopic enrichment studies that were consistent new peaks compared with the controls. In contrast to LipK (14), the stereochemistry of the CapH product was not previously with L-Thr as the source of the carbon and nitrogen atoms of the assigned (11). Therefore, two additional stereoisomers of GlyU— carboxamide of CarU and, significantly, that the C-N bond of ′ ′ ′ ′ — GlyU remains intact during the transformation to CarU (11). (5 R,6 S)and(5S,6 R) that could hypothetically arise from the transaldolase reaction (14) were tested for activity with The totality of the results suggested that the remaining steps to Cap15. We have detailed the stereocontrolled synthesis and furnish CarU minimally involve (i) decarboxylation of GlyU, (ii) analytic characterization of these two diastereomers elsewhere incorporation of an oxygen atom via a hydratase or oxygenase (15–17). Once again, however, no new peaks were observed activity, and (iii) oxidation to the carboxamide. In lieu of bio- with Cap15. chemical precedence of PLP-dependent enzymes, it seemed We next explored a variety of reaction conditions including reasonable that ORF15 might serve as the catalyst for the de- different pH and buffer systems. When reactions were per- carboxylation step, despite inference from sequence analysis. formed in phosphate buffer with the supposedly unnatural (5′ “ ” In our attempt to delineate the remaining steps that trim GlyU S,6′R)-GlyU isomer, a new peak was observed by HPLC, and to CarU, we now report the function of ORF15, herein named formation of the new peak was dependent upon the inclusion of Cap15. As anticipated, Cap15 catalyzes decarboxylation of GlyU, Cap15 and PLP (Fig. 2A). A trace amount of the new peak but also has an unexpected oxygenase activity and is identified as the appeared following reactions with Cap15(K230A) (Fig. 2A). No sole catalyst responsible for the complete conversion of GlyU to the new peaks were detected using the (5′S,6′S)or(5′R,6′S) isomers signature CarU nucleoside of the capuramycin-type metabolites. of GlyU as potential substrates under the phosphate-buffered Specifically, the data are consistent with the functional assignment conditions (SI Appendix, Fig. S5). The new peak was analyzed + of Cap15 as a PLP-dependent (5′S,6′R)-GlyU monooxygenase- by LC-MS yielding an (M + H) ion at m/z = 288.0, unexpectedly

decarboxylase. Additional biochemical studies have enabled us to consistent with the molecular formula for CarU [expected BIOCHEMISTRY + + propose a mechanistic framework for this PLP-dependent enzyme- (M + H) ion at m/z = 288.1 and (M + Na) ion at m/z = 310.0].

Huang et al. PNAS | January 30, 2018 | vol. 115 | no. 5 | 975 Downloaded by guest on September 23, 2021 (SI Appendix, Fig. S3B), consistent with Cap15 functioning as an oxygenase. We subsequently probed the origin of the oxygen atom of the carboxamide of CarU, which hypothetically could be derived from phosphate, O2,orH2O. Three independent reac- 18 18 tions were performed with isotopically enriched H3P O4,H2 O, 18 or O2, and CarU production was monitored by LC-MS (Fig. 2B). + An (M + H) ion at m/z = 290.1 was detected only in the presence 18 of O2, which corresponds to a 2-amu increase compared with unlabeled CarU. An estimated 93% enrichment was calculated based on peak intensities under these conditions. Finally, hydro- gen peroxide was not detected under optimized reaction condi- tions. Thus, the data are consistent with the classification of Cap15 as a monooxygenase and also suggest that phosphate does not participate directly in the chemistry of the Cap15 reaction. The identification of CarU as the product suggested that CO2 was concomitantly formed during the reaction. To confirm this, we used a commercial enzyme-linked bicarbonate detection system for in situ detection of CO2. Using reactions without PLP or phosphate as controls, CO2 was confirmed as the second product of the Cap15-catalyzed reaction (SI Appendix, Fig. S3C). Contrastingly, reactions under anaerobic conditions did not produce detectable CO2. Furthermore, reactions with Cap15(K230A) resulted in only trace amounts of CO2. In total, the data are consistent with the functional assignment of Cap15 as a PLP- dependent (5′S,6′R)-GlyU:O2 monooxygenase-decarboxylase that generates carboxamide-containing CarU and CO2.

Biochemical Properties. Cap15 was active only in phosphate buffer, wherein the pH profile resembled a bell-shaped curve with op- timal activity at pH 7.5 (Fig. 3A). The specific activity of − Cap15 was (5.2 ± 0.2) × 10 3 μmol/min/mg under the optimized conditions. The specific activity with Cap15(K230A) was (2.4 ± − 0.1) × 10 4 μmol/min/mg, an approximately 20-fold decrease compared with the wild-type enzyme. Using HPLC to detect CarU formation, single-substrate kinetic analysis with variable (5′S,6′R)-GlyU revealed Michaelis–Menten kinetics yielding a 1 −1 −1 Km = (56 ± 7) × 10 μMandkcat = (9.3 ± 0.5) × 10 min (Fig. 3B). Increasing the concentration of phosphate from 50 mM to 1 M had little impact with respect to the overall catalytic efficiency 1 −1 −1 [Km = (42 ± 4) × 10 μMandkcat = (6.2 ± 0.2) × 10 min ] (SI Appendix, Fig. S13). Furthermore, nearly identical kinetic parameters were calculated when activity was measured by CO2 detection instead of CarU detection by HPLC (Fig. 3B). As previously noted, the activity of Cap15 was stereoselective for the (5′S,6′R)-GlyU diastereomer. Thus, the reaction cata- lyzed by CapH was re-examined to identify the stereochemistry Fig. 2. Biochemical characterization of Cap15. (A) HPLC traces of the re- of the GlyU product. Initially, a one-pot reaction with CapH and action catalyzed by Cap15 using substrate (5′S,6′R)-GlyU with (i) exclusion of ′ ′ ′ Cap15 starting from U5 A and L-Thr was analyzed by HPLC, enzyme; (ii) complete reaction mixture containing phosphate, (5 S,6 R)-GlyU, SI Ap- O , PLP, and Cap15; (iii) exclusion of PLP; (iv) substitution of Cap15(K230A) revealing the formation of a peak coeluting with GlyU ( 2 pendix, Fig. S14). However, no CarU was observed, suggesting for the wild-type enzyme; (v) exclusion of O2; and (vi) synthetic CarU. A260, absorbance at 260 nm. (B) LC-MS analysis of the CarU product following that the CapH product—presumably (5′S,6′S)-GlyU—is not a Cap15-catalyzed reactions in the presence of the indicated isotopically la- substrate for Cap15. Subsequently, the stereochemistry of the beled molecule. Data shown are representative of duplicates. RA, relative CapH product was directly assessed. As described elsewhere abundance. (14), phosgene modification of the β–hydroxy-amino acid of GlyU enabled the analytical separation of the three synthetic diastereomers in hand. Following the CapH reaction using syn- To simplify product identification, CarU was synthesized and thetic U5′A and L-Thr as substrates, the phosgene-modified purified, and authentic CarU coeluted with the Cap15 product GlyU coeluted with authentic, phosgene-modified (5′S,6′S)- and had identical mass and NMR spectroscopic properties (Fig. GlyU (Fig. 3C). Modification of the LipK product as a control – 2A and SI Appendix,Figs.S6S11). HPLC analysis of the confirmed this result (14). Thus, CapH stereospecifically gen- Cap15 reaction revealed the enzyme was inactive under anaerobic erates (5′S,6′S)-GlyU, which must undergo epimerization before conditions (Fig. 2A) and inclusion of EDTA had no effect (SI the Cap15-catalyzed reaction. Appendix,Fig.S12). Thus, Cap15 catalyzes a PLP-, phosphate-, and O2-dependent conversion of (5′S,6′R)-GlyU to CarU. Initiatory Mechanistic Analysis. The K230A mutation of Cap15 did The unanticipated requirement of O2 and phosphate for not abolish activity as initially predicted, and thus site-directed Cap15 activity led us to more closely examine their role in the mutants of the two remaining conserved Lys residues (Lys265 and reaction. Using a sealed vessel and monitoring the reaction with Lys303) were prepared, and the mutant proteins analyzed for 262 an O2 electrode, dissolved O2 was steadily consumed over time activity. Cap15-Lys was also targeted for mutagenesis due to

976 | www.pnas.org/cgi/doi/10.1073/pnas.1718667115 Huang et al. Downloaded by guest on September 23, 2021 Fig. 3. Activity analysis of Cap15 and CapH. (A) Dependence of Cap15 activity on buffer and pH. (B) Single-substrate kinetic analysis with variable (5′S,6′R)-

GlyU by detection of CarU product by HPLC (○) or by detection of coproduct CO2 (■). (C) HPLC traces of (i) phosgene-modified, synthetic (5′S,6′R)-GlyU (●); (ii) phosgene-modified, synthetic (5′S,6′S)-GlyU (♦); (iii) phosgene-modified product of CapH-catalyzed reaction; and (iv) negative control without CapH.

the importance of the corresponding Arg residue in AaSelA (SI Cap15(K230A) was essentially identical to free PLP. The λmax = Appendix, Fig. S1). The three additional Lys → Ala mutant 411 nm for Cap15(K265A) did not shift but increased in intensity proteins were soluble when expressed in E. coli (SI Appendix,Fig. following the addition of PLP (SI Appendix, Fig. S16B). Upon S3A), and activity analysis by HPLC revealed that Cap15(K303A) addition of excess (5′S,6′R)-GlyU to the wild-type Cap15-PLP retained activity comparable to the wild-type enzyme (SI Appendix, solution, a red shift was observed to λmax = 415 nm (Fig. 4A, Fig. S15). Contrastingly, the K262A and K265A mutant proteins trace IV). Cap15(K230A) and Cap15(K303A), both active as did not produce detectable levels of CarU. catalysts, had similar spectral shifts (SI Appendix, Fig. S16C). In The UV/Vis absorption spectra for the wild-type and mutant contrast, the UV/Vis spectra for Cap15(K262A) and Cap15(K265A) proteins were examined with the goal of establishing a mecha- did not change (SI Appendix,Fig.S16C). As expected, nonsubstrates nistic framework for Cap15 (2, 18, 19). Wild-type Cap15 and the (5′S,6′S)-GlyU and (5′R,6′S)-GlyU did not significantly alter the mutant variants did not have an obvious absorbance at wave- spectral profile when added to the wild-type Cap15-PLP mixture lengths greater than 300 nm, consistent with the production of (SI Appendix,Fig.S17). Closer examination of the Cap15 UV/Vis the recombinant enzyme in an apo-form (Fig. 4A, trace I, and SI spectrum revealed a second λmax at 503 nm, which appeared im- Appendix, Fig. S16A). The lone exception was Cap15(K265A), mediately following the addition of (5′S,6′R)-GlyU and remained λ = which displayed a max 411 nm that is consistent with some nearly constant over the course of 24 min (Fig. 4B, trace V). In protein copurifying with PLP as the internal aldimine (SI Ap- contrast to the other mutant proteins, only Cap15(K303A) displayed pendix, Fig. S16A). Identical results were obtained when in- the identical λmax at 503 nm (SI Appendix,Fig.S16D). cluding excess PLP before protein purification by affinity chromatography. Following the addition of substoichiometric Discussion PLP, which has a λmax at 388 nm representing the aldehyde form The functional assignment of Cap15 has revealed unprecedented (Fig. 4A, trace II), a subtle red shift to λmax at 391 nm was de- enzyme chemistry that involves PLP as an oxygenase cofactor, an tected for wild-type Cap15 (Fig. 4A, trace III), Cap15(K262A), unusual role for an already well-known and highly versatile co- and Cap15(K303A) (SI Appendix, Fig. S16B); the spectrum for factor. Several enzymes involved in primary metabolism that

Fig. 4. Mechanism of Cap15. (A) UV/Vis spectra of PLP in phosphate pH 7.5 (dotted line); Cap15 before (dashed line) and after (black solid line) the addition of PLP; and the ternary complex after addition of (5′S,6′R)-GlyU (rainbow lines). (B) UV/Vis spectra for Cap15-PLP (black solid line) and ternary complex BIOCHEMISTRY (rainbow lines) at longer wavelength revealing the λmax at 503 nm. Spectral datasets were zeroed at 600 nm. (C) Proposed mechanism for Cap15. The roman numerals under the chemical species are proposed in part based on interpretation of the spectra highlighted in A and B.

Huang et al. PNAS | January 30, 2018 | vol. 115 | no. 5 | 977 Downloaded by guest on September 23, 2021 generate carbanionic intermediates have previously been reported aminotransferase (28) and 1-aminocyclopropane-1-carboxylate to catalyze off-pathway side reactions—so-called paracatalytic synthase (29), among others (2, 19), have revealed an internal reactions—that involve oxidation of the carbanionic interme- aldimine that—depending upon the orientation and protonation diate with O2 (20, 21). Given that most members of the PLP- state of PLP in the active site—can display a UV/Vis spectrum dependent transferase superfamily are characterized by their with a λmax ranging from 390 nm (corresponding to an unproto- ability to stabilize a carbanion through electron delocaliza- nated imine) to 420 nm (corresponding to a protonated imine). tion within PLP, it is not surprising that five distinct PLP- Additionally, mutational analysis of Cap15 revealed that the in- dependent enzymes are included in the paracatalytic reaction active mutant Cap15(K265A) displays a spectrum with a λmax of list (20). These five PLP-dependent enzymes use an amino 411 nm following purification and addition of exogenous PLP, acid substrate to catalyze oxidative deamination in addition which is consistent with formation of a protonated internal aldi- to decarboxylation, the former of which was not detected with mine (30, 31). In lieu of the literature precedent along with these Cap15. For three of these PLP-dependent enzyme “paracatalysts,” data, we propose a mechanism wherein Cap15 does indeed form a H2O2 is stoichiometrically produced, and thus they are classified protonated internal aldimine (III) with an unidentified Lys that is 265 as oxidases (20, 22–25). The fate of the oxygen atoms for the characterized by a λmax of 391 nm (Fig. 4B). Mutation of Lys is other two paracatalysts, however, remains unknown. proposed to promote orientation of the internal aldimine to favor In contrast to PLP-dependent enzymes that catalyze para- protonation, hence displaying the more traditional spectrum as- catalytic reactions, a few enzymes have recently been reported to sociated with PLP-dependent enzymes. Regardless, the red shift catalyze PLP-dependent oxidations as their primary function (3, 4, to λmax at 416 nm following the addition of (5′S,6′R)-GlyU to the 26, 27). Ind4, involved in indolymycin biosynthesis, was shown to Cap15-PLP complex strongly suggests the formation of an exter- catalyze tandem 2,3- and 4,5-dehydrogenation of L-Arg, concomi- nal aldimine (IV) that undergoes Cα-deprotonation to form a tantly reducing O2 to H2O2 (3). CcdF, involved in celesticin bio- resonance-stabilized quininoid (V), which is characterized by the synthesis, was shown to catalyze an oxidative decarboxylation and appearance of a second λmax at 503 nm in the spectrum for wild- deamination of an α–amino-acid component of a celesticin pre- type Cap15 and Cap15(K303A). Similar to the results with MppP cursor, also concomitantly reducing O2 to H2O2 (26). Thus, both and Ind4 (3, 4), the quininoid appears to reach a steady state Ind4 and CcdF are bona fide PLP-dependent oxidases. Contrastingly, during the reaction that would suggest that downstream chemistry MppP involved in L-enduracidine biosynthesis was shown to cata- is rate-limiting (2). lyze an O2 and PLP-dependent α-deamination and 4-hydroxylation The most intriguing aspect of the Cap15 reaction is the oxi- of L-Arg, the latter suggesting that this enzyme is an oxygenase (4). dative steps that follow aldimine formation. Other enzymes that However, neither the fate of the oxygen atoms of O2 nor the origin of catalyze oxidative decarboxylation of α-amino acids to form the oxygen atom in the hydroxylated product was determined, and carboxamides—namely Trp (32), Lys (33), Arg (34), and Phe thus the enzyme classification of MppP is not clear. Our results de- (35) 2-monooxygenases–require flavin adenine dinucleotide finitively establish the use of PLP as a redox cofactor that enables (FAD) to initiate electron transfer to dioxygen (36). For Trp-2- Cap15 to function as a true monooxygenase, incorporating one monooxygenase, the best characterized of the carboxamide- OatomofO2 directly into the carboxamide-containing product forming flavoproteins, the reaction begins by hydride transfer CarU. Interestingly, a similar PLP-dependent, α–amino-acid-to- from Trp to FAD to generate an imine and reduced FAD, re- carboxamide transformation has been proposed during pyoverdine spectively (32). The reduced FAD subsequently donates an biosynthesis, although the activity of the probable catalyst (PvdN) electron to O2 to yield a caged radical pair that, following spin remains to be demonstrated in vitro (27). inversion, recombines to form a C(4a)-hydroperoxide interme- Bioinformatically, Cap15 has primary sequence homology to diate (34). Contrastingly, the oxidative steps of the Cap15-cata- bacterial proteins annotated as SelA, which is a fold-type I PLP- lyzed reaction do not require a flavin cofactor, nor does it appear dependent enzyme that catalyzes the transformation of seryl-tRNA that a redox active metal cofactor is involved. Consequently, we (Sec) to selenocysteinyl-tRNA(Sec) using selenophosphate as the propose a hydroperoxide mechanism for Cap15 (Fig. 4B), selenium donor (13). The discovery that the Cap15 reaction not wherein the carbanion resonance form of the quininoid (V) only requires O2 but also depends on phosphate, in this case the serves a role that is comparable to the reduced FAD for Trp-2- standard oxygenated variant, gave rise to the possibility that the monooxygenase. Thus, an electron is first transferred from the O atom incorporated into CarU could originate directly from carbanion to O2 to form a resonance-stabilized GlyU-PLP rad- phosphate in a reaction analogous to that catalyzed by SelA. ical and superoxide, the latter of which can undergo protonation However, the isotopic enrichment studies with 18O-labeled and rebound with the carbon-centered radical to form the hy- 18 phosphate, compared with O-labeled O2, clearly revealed that droperoxide species. Subsequently, decarboxylation occurs with this was not the case, thus leaving the role of phosphate in the elimination of the distal hydroperoxyl O atom in the form of Cap15-catalyzed reaction unknown. Interestingly, the dioxyge- water to generate a CarU-PLP aldimine that is ultimately hy- nase CapA, which initiates the CarU biosynthetic pathway, drolyzed to release CarU. Although it remains possible that the produces phosphate via an unusual oxidative dephosphorylation initial carbanion is generated by decarboxylation instead of mechanism that could potentially participate as a feed-forward deprotonation of the aldimine, we were unable to detect CO2 allosteric modulator (11). Preliminary analysis suggests that under anaerobic conditions, thus suggesting that O2 reduction Cap15 does in fact differentially oligomerize in the presence of precedes decarboxylation. phosphate (SI Appendix, Fig. S18), although ongoing kinetic and In addition to the unusual role of PLP as a redox cofactor, the structural studies will be essential to clarify the quaternary structure finding that Cap15 is stereospecific for the (5′S,6′R)-diastereomer and role of phosphate. of GlyU was unexpected. This led us to reexamine the stereo- Wild-type Cap15 (I) does not copurify with a detectable amount chemical outcome of the transaldolase CapH. Although it is of PLP (Fig. 4B) and is inactive without an exogenous supply of mechanistically feasible for CapH to produce (5′S,6′R)-GlyU, the PLP. Furthermore, the addition of PLP to Cap15 (III) resulted in product was clearly identified as (5′S,6′R)-GlyU upon compari- a UV/Vis spectrum that is nearly indistinguishable from the al- sons with synthetic standards and the product of the well-char- dehyde form of PLP (II), which is in contrast to most other PLP- acterized transaldolase LipK (14). Thus, the discovery of Cap15 dependent enzymes that display a significant red shift (λmax of activity—despite filling a major gap regarding the biosynthetic 410–440 nm) that is indicative of an internal aldimine (19). As a mechanism of CarU—introduces another biochemical step in the result, we initially considered a mechanism that bypasses the in- pathway and suggests the involvement of an additional, un- ternal aldimine. However, studies with PLP-dependent aspartate identified epimerase. Intriguingly, a putative NAD-dependent

978 | www.pnas.org/cgi/doi/10.1073/pnas.1718667115 Huang et al. Downloaded by guest on September 23, 2021 NDP-hexose epimerase is among the few remaining unassigned enzymes per genome with as-of-yet-unknown function hints at proteins within the gene cluster; however, other possibilities can- the possibility that other PLP-dependent oxygenases not unlike not be excluded at this time. Cap15 remain to be discovered. In summary, we have provided evidence to support the func- tional assignment of Cap15 as a PLP-dependent (5′S,6′R)-GlyU: Materials and Methods O2 monooxygenase-decarboxylase that generates the carboxamide Cloning, mutatgenesis, heterologous production, and activity analysis for functionality of CarU, the signature nucleoside core of the Cap15 were performed using standard procedures and are described in the SI capuramycins. Generally, enzymes known to activate O2 do so by Appendix, SI Materials and Methods. Reactions were monitored by C18 re- employing a flavin or transition metal cofactor or, in some in- verse phase HPLC, LC-MS, a Carbon Dioxide Enzymatic Assay kit (Diazyme stances, are substrate-assisted (also known as cofactor indepen- Laboratories), and a Profiling Oxygen Microsensor PM-PSt7 equipped with dent). Indeed, enzymes that catalyze a comparable α-amino Microx 4 oxygen meter (PreSens Precision Sensing), and UV-visible spectros- acid→carboxamide conversion require a reduced FAD to achieve copy was performed with a Shimadzu UV/Vis-1800 spectrophotometer. The this chemical outcome. Characterization of Cap15 now provides a synthesis of (5′R,6′S)-GlyU, (5′S,6′R)-GlyU, and U5′A was previously described – ′ ′ definitive example of an enzyme whose native function requires (14 17). The syntheses and analytical analyses of (5 S,6 S)-GlyU and CarU are PLP as an oxygenase cofactor. With the mechanistic framework described in the SI Appendix, SI Materials and Methods. provided here, it is now possible to compare the role of the flavin ACKNOWLEDGMENTS. We thank Dr. Koichi Nonaka (Daiichi-Sankyo Co.) for and PLP cofactors as it relates to the oxidative decarboxylation of intellectual contributions and for providing strains and cosmids. This work amino acids to produce the respective carboxamide-containing was supported in part by National Institutes of Health Grants AI128862 and products. Furthermore, the abundance of predicted PLP-dependent AI087849 (to S.G.V.L.).

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