Dissecting the low catalytic capability of flavin-dependent halogenases
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.016004
ISSN1083-351X
AutoresAisaraphon Phintha, Kridsadakorn Prakinee, A. Jaruwat, Narin Lawan, Surawit Visitsatthawong, Chadaporn Kantiwiriyawanitch, Warangkhana Songsungthong, Duangthip Trisrivirat, Pirom Chenprakhon, Adrian J. Mulholland, Karl‐Heinz van Pée, P. Chitnumsub, Pimchai Chaiyen,
Tópico(s)Enzyme Structure and Function
ResumoAlthough flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I−, Br−, and Cl− but not F− to form C4a-hydroxyflavin and HOX. Our experiments revealed that I− reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications. Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I−, Br−, and Cl− but not F− to form C4a-hydroxyflavin and HOX. Our experiments revealed that I− reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications. Halogenation is extremely important for the agricultural, pharmaceutical, and chemical industries (1Jeschke P. The unique role of halogen substituents in the design of modern agrochemicals.Pest Manag. 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Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH.Biochemistry. 2006; 45: 7904-7912Crossref PubMed Scopus (86) Google Scholar), hypohalous acid (HOX) (hypobromous acid [HOBr] and hypochlorous acid [HOCl]). HOX is thought to be transferred to a second active site to react with tryptophan (27Yeh E. Blasiak L.C. Koglin A. Drennan C.L. Walsh C.T. Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases.Biochemistry. 2007; 46: 1284-1292Crossref PubMed Scopus (152) Google Scholar, 28Yeh E. Cole L.J. Barr E.W. Bollinger J.M. Ballou D.P. Walsh C.T. Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH.Biochemistry. 2006; 45: 7904-7912Crossref PubMed Scopus (86) Google Scholar, 29Flecks S. Patallo E.P. Zhu X. Ernyei A.J. Seifert G. Schneider A. Dong C. Naismith J.H. van Pée K.-H. 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Haupt C. van Pée K.-H. Naismith J.H. Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination.Science. 2005; 309: 2216-2219Crossref PubMed Scopus (264) Google Scholar, 32Karabencheva-Christova T.G. Torras J. Mulholland A.J. Lodola A. Christov C.Z. Mechanistic insights into the reaction of chlorination of tryptophan catalyzed by tryptophan 7-halogenase.Sci. Rep. 2017; 7: 17395Crossref PubMed Scopus (14) Google Scholar). Interestingly, none of the studies since then have investigated the function of the conserved Lys. None of the previous studies has also investigated the mechanism of HOX formation and determined the factors controlling HOX production. Most FDHs are inefficient because their turnovers are quite slow (kcat = 0.5–3 min−1), and they can be easily inactivated (26Andorfer M.C. Lewis J.C. Understanding and improving the activity of flavin-dependent halogenases via random and targeted mutagenesis.Annu. Rev. Biochem. 2018; 87: 159-185Crossref PubMed Scopus (27) Google Scholar). Despite gram scale synthesis of halogenated tryptophan product was reported for the reaction of RebH, full conversion was obtained only after 8 days (33Frese M. Sewald N. Enzymatic halogenation of tryptophan on a gram scale.Angew. Chem. Int. Ed. Engl. 2015; 54: 298-301Crossref PubMed Scopus (90) Google Scholar), which is too slow to be accepted for a real industrial process. Therefore, kinetic and structural investigation to understand mechanistic features controlling their halogenation are necessary for future improvement of FDHs for industrial applications. Tryptophan 6-halogenase or Thal from Streptomyces albogriseolus (34Moritzer A.-C. Niemann H.H. Binding of FAD and tryptophan to the tryptophan 6-halogenase Thal is negatively coupled.Protein Sci. 2019; 28: 2112-2118Crossref PubMed Scopus (10) Google Scholar, 35Moritzer A.-C. Minges H. Prior T. Frese M. Sewald N. Niemann H.H. Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal.J. Biol. Chem. 2019; 294: 2529-2542Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) represents an interesting FDH system for investigating the mechanisms of HOX formation and factors controlling halogenation. As Thal catalyzes tryptophan halogenation at the 6-position, not 7-position as in RebH or PrnA, the results would also contribute to the understanding of a mechanism related to halogenation of tryptophan at the 6-position. Recently, crystal structures of Thal were solved and the results showed that FAD and tryptophan substrates could not be co-crystallized (34Moritzer A.-C. Niemann H.H. Binding of FAD and tryptophan to the tryptophan 6-halogenase Thal is negatively coupled.Protein Sci. 2019; 28: 2112-2118Crossref PubMed Scopus (10) Google Scholar). This feature is different from other tryptophan halogenases such as RebH (36Bitto E. Huang Y. Bingman C.A. Singh S. Thorson J.S. Phillips Jr., G.N. The structure of flavin-dependent tryptophan 7-halogenase RebH.Proteins. 2008; 70: 289-293Crossref PubMed Scopus (67) Google Scholar), tryptophan 5-halogenase (PyrH) (37Zhu X. De Laurentis W. Leang K. Herrmann J. Ihlefeld K. van Pée K.-H. Naismith J.H. Structural insights into regioselectivity in the enzymatic chlorination of tryptophan.J. Mol. Biol. 2009; 391: 74-85Crossref PubMed Scopus (85) Google Scholar) and PrnA (15Dong C. Flecks S. Unversucht S. Haupt C. van Pée K.-H. Naismith J.H. Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination.Science. 2005; 309: 2216-2219Crossref PubMed Scopus (264) Google Scholar) which could be co-crystallized with both FAD and tryptophan. This suggests that protein dynamics between the flavin and substrate binding sites of Thal are strongly linked. Therefore, we set out to investigate the mechanism of HOX formation and its role in regulating the Thal reaction and used Thal as a model for exploring the factors contributing to the catalytic inefficiency of FDHs in general. In this report, the reaction of Thal was investigated with regard to four main mechanistic issues to unravel why the reactions of FDHs are not very efficient. (A) The mechanism of HOX formation was investigated using transient kinetics (stopped-flow absorbance and fluorescence and rapid-quench flow techniques) in combination with quantum/mechanics molecular mechanics (QM/MM) computational calculations. (B) The cause of the low product coupling ratio was investigated by measuring the leakage of the HOX intermediate using an organic substrate (D-luciferin) as a halogenating target to detect free HOX. (C) The functional role of the conserved Lys was investigated using site-saturation mutagenesis studies along with detection of free HOX formation and theoretical pKa calculations. Unlike the mechanism previously proposed, our results do not support formation of chloramine or bromamine intermediates but rather suggest that the conserved Lys likely protonates the HOX in the halogenation reaction. (D) Results from transient kinetics, newly crystallized structures of Thal:FADH− and Thal:FAD:AMP and molecular dynamics (MD) simulations could be used to explain the cause for formation of the dead-end inactive Thal:FADH-∗ complex. To explore whether Thal represents a halogenase which can catalyze a reasonable halogenation reaction, multiple turnover reactions of Thal were carried out and compared with the reactions of PrnA and PyrH. The products from the Thal reaction were characterized by NMR spectroscopy (Fig. S2C). The results indicated that among the three halogenases investigated, Thal was the fastest halogenase (Fig. S2A). These data also clearly imply that most of the halogenases are not very efficient enzymes which agrees with previous data reporting that FDHs suffer from having low kcat values (26Andorfer M.C. Lewis J.C. Understanding and improving the activity of flavin-dependent halogenases via random and targeted mutagenesis.Annu. Rev. Biochem. 2018; 87: 159-185Crossref PubMed Scopus (27) Google Scholar). Therefore, understanding the cause of catalytic inefficiency in Thal as a representative of FDHs would contribute to identification of the bottleneck in the reaction of FDHs in general. The percentage of product halogenation in the Thal reaction was measured from the consumption of the substrate (tryptophan) in single turnover reactions carried out in a rapid-quench flow instrument (Experimental Procedures). The reaction of Thal was carried out using FADH− as a limiting reagent (Fig. 1). After the first turnover was completed (data collected at 400 s), we found that only about 30% of the tryptophan was halogenated (data not shown). The results indicate that the coupling ratio (product produced per substrate consumed) is quite low, which is likely one of the factors explaining the catalytic inefficiency in Thal and in FDHs in general. Therefore, we further explored the fates of each reaction intermediate after C4aOOH-FAD is formed. A stopped-flow experiment was carried out under conditions in which C4aOOH-FAD was fully formed (details described in Figs. S5 and S6). In the absence of halide, mixing of an anaerobic solution of FADH− with an aerobic solution of Thal resulted in reactions with kinetic traces showing two phases (Fig. 2). The first phase (0.02–0.15 s) showed an increase in absorbance at 380 nm with almost no change at 450 nm. The second phase (0.15–100 s) showed a decrease in absorbance at 380 nm which was concomitant with an increase in absorbance at 450 nm. Kinetic traces of the first phase and second phase represent C4aOOH-FAD formation (kobs of 16.52 s−1) and decay (kobs of 0.41 s−1) to form hydrogen peroxide (H2O2) and FAD, respectively (Fig. 2). The kinetics of C4aOOH-FAD formation in the presence of 10 mM Br− or Cl− (the condition in which HOBr or HOCl was formed maximally, as explained in detail in the next section) was the same as that in the absence of Br− or Cl− (Fig. 2), but the kinetics of the reaction after C4aOOH-FAD formation was different. The reaction in the presence of halide ions showed three phases. The first phase (0.02–0.15 s) showed an increase in absorbance at 380 nm. The second (0.15–2.5 s) and third phases (2.5–100 s) showed a decrease in absorbance at 380 nm and an increase in absorbance at 450 nm. The kinetics of the second phase of both reactions (kobs of 1.6 s−1) were similar to that in the absence of halide, indicating that this phase was the uncoupling path to elimination of H2O2 without forming HOBr or HOCl. As the amplitude of this phase (absorbance 450 nm) in the presence of Br− and Cl− was about 20% and 55%, respectively, the results indicate that the uncoupling path is more prominent in the presence of Cl− compared with Br− and that C4aOOH-FAD reacts more efficiently with Br− than Cl−. The kinetics of the third phase was clearly different from the reaction in the absence of the halide ions. This phase was later identified as the step of C4a-hydroxyflavin (C4aOH-FAD) decay after HOX formation (see the following results in the next section). These data indicate that even under saturating amounts of halide, the reaction of Thal still proceeds through an uncoupling path (no formation of HOX) with a frequency of about 20% and 55% in the case of Br− and Cl−, respectively. The data also indicate that 80% of the C4aOOH-FAD intermediate can react with Br− to form HOBr. However, our results in the first section (halogenation activity of Thal) indicate that only 30% of the tryptophans were brominated, implying that not all of the HOBr formed reacts with tryptophan. We therefore investigated the mechanisms of HOX formation by FDHs and examined their control of HOX reactivity. Because the reaction of halide ions with C4aOOH-FAD is crucial for the catalysis of FDHs and this reaction is not well understood, we investigated the reactivity of halide ions with Thal and other tryptophan halogenases (PrnA and PyrH) by monitoring the kinetics of its formation. To monitor the kinetics of HOX formation, we monitored the transitions from C4aOOH-FAD to C4aOH-FAD and HOX (Fig. 3). C4aOH-FAD is a common intermediate in the reactions of flavin-dependent monooxygenases (28Yeh E. Cole L.J. Barr E.W. Bollinger J.M. Ballou D.P. Walsh C.T. 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Therefore, we carried out similar experiments as in the previous section but instead we measured the appearance of highly fluorescent species using excitation wavelengths (Ex) of 380 nm and 450 nm and monitoring emission wavelength at > 495 nm. Results in Figure 4 show a large increase in fluorescence during 0.15 to 2.5 s (corresponding to an observed rate constant of 1.5 s−1) in the presence of Cl− when using Ex at 380 nm (blue solid line). This fluorescent species decayed during 2.5 to 100 s (corresponding to an observed rate constant of 0.073 s−1). The data clearly indicate that the fluorescence decay occurred simultaneously with the third phase of Figure 2 in which the absorbance at 380 nm decreased and the absorbance at 450 nm increased. The data in Figures 2 and 4 indicate that in the presence of Cl− after C4aOOH-FAD was formed, and the next step was formation of a highly fluorescent C4aOH-FAD species which eventually eliminates H2O to form oxidized FAD (Fig. 3). The conversion of C4aOOH-F
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