An Acyl Transfer Reaction Catalyzed by an Epimerase MarH - ACS

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An Acyl Transfer Reaction Catalyzed by an Epimerase MarH Mo Han,†,‡ Haixing Yin,§,‡ Yi Zou,† Nelson L. Brock,† Tingting Huang,† Zixin Deng,† Yiwen Chu,*,§ and Shuangjun Lin*,† †

State Key Laboratory of Microbial Metabolism, Joint International Laboratory on Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China § Sichuan Industrial Institute of Antibiotics, Chengdu University, 168 Huaguan Road, Chengdu 610052, China S Supporting Information *

ABSTRACT: MarH, a small protein (129 amino acids) belonging to the cupin superfamily, was previously characterized as an epimerase involved in the (2S,3S)-β-methyltryptophan formation in the maremycin biosynthesis. Here, MarH was discovered to act as an acyltransferase that can catalyze the 3-O-acylation of chloramphenicol. Furthermore, MarH can catalyze N-acylation of deacylated chloramphenicol analogue thereby activating them for 3-O-acylation. By systematic site-directed mutagenesis, H64 was revealed as a potential catalytic base that deprotonates the acyl acceptor substrate. Nucleophilic attack at the carbonyl carbon of the acyl donor then gives the acylation product. KEYWORDS: cupin protein, MarH, multifunctional enzyme, acyltransferase, chloramphenicol acetyltransferase

M

expression plasmid pET28a-marH (hereafter named as strain-I) and the control plasmid pET28a (named as strain-II) were, respectively, cultured in LB medium containing both kanamycin and Cml (Supporting Information, SI). After fermentation, the supernatants were analyzed by high-performance liquid chromatography−mass spectrometry (HPLC-MS). Three peaks showed the same UV absorption as Cml, but their relative intensities differed in the strains. In particular, 4 was the dominant product in strain-I while 2 was the major product in strain-II (Figure 1A). High-resolution mass (HR-MS) analyses suggested that they were the acetylation products of 1 (Table S2 and Figure S14). The three compounds were purified, and comprehensive NMR analysis confirmed 2−4 to be 3-O-acetyl Cml, 1-O-acetyl Cml and 1,3-O-diacetyl Cml, respectively (Figure S15−20). Thus, we presumed that MarH might exhibit the CAT activity in addition to its epimerase activity. To verify this speculation, MarH was overexpressed in E. coli BL21(DE3) instead of E. coli BL21(DE3)pLysS to avoid contamination of CATs. The in vitro CAT activity assays (SI) proved that the purified MarH efficiently catalyzed the acetyl transfer from acetyl coenzyme A (Ac-CoA, 5) to Cml, generating both monoacetylated Cml 2 and 3, and diacetylated Cml 4 (Figure 1B). The time-dependent assays showed that initially only 2 was produced, followed by production of 3, and that 4 appeared last (Figure S1). This result implicated that 3 was nonenzymatically generated from 2, which was confirmed by incubation of 2 in Tris-HCl buffer at 30 °C (Figure S2A). The nonenzymatic transformation may take place via a six-membered cyclic

ost enzymes are special proteins produced by organisms that can catalyze diverse chemical reactions in life process. Due to their versatility, chemo-, regio-, enantioselectivity, and mild reaction conditions,1 they are explored as catalysts in many chemical industries.2 The selectivity and specificity of enzymes are evidenced by the induced-fit toward their substrates: the binding of substrates induces a conformational change of an enzyme. The conformational change operates like a molecular switch to recognize correct substrates.3 A growing number of enzymes with substrate ambiguity4 and multifunctional enzymes with different domains, such as fatty acid synthetases,5 polyketide synthases,6 nonribosomal peptide synthetases,7 and many others8 have been reported. In addition, single-domain enzymes with catalytic promiscuity are also known,9 but most of them catalyze similar or sequential reactions.10 MarH (AHJ60977) belongs to the cupin superfamily that is widely found in prokaryotic and eukaryotic organisms.11 This versatile superfamily includes metal-independent epimerases12 and metal-dependent enzymes such as dioxygenases, 13 decarboxylases,14 hydroxylases,15 and more.16 Most cupin proteins catalyze a single reaction. An exception is the barley germin that can act as both oxalate oxidase and manganese superoxide dismutase.17 Previously, we have shown that MarH catalyzes the epimerization of (R)-β-methylindolepyruvate in the biosynthesis of (2S,3S)-β-methyltryptophan from Ltryptophan.18 Here, we report that MarH can also catalyze the acylation of chloramphenicol (Cml, 1) even though it shows no homology to Cml acetyltransferases (CATs). When MarH was overexpressed in E. coli BL21(DE3) pLysS in Luria−Bertani (LB) medium supplemented with Cml, we accidently discovered that MarH could catalyze the acetylation of Cml. The strain E. coli BL21(DE3)pLysS harboring the © 2015 American Chemical Society

Received: September 30, 2015 Revised: November 26, 2015 Published: December 30, 2015 788

DOI: 10.1021/acscatal.5b02198 ACS Catal. 2016, 6, 788−792

Letter

ACS Catalysis

Figure 1. Activity and substrate specificity of MarH for acyl-donors. (A) HPLC profiles of fermentation extracts of strain-I (i) and strain-II (ii). (B) HPLC profiles of acylation of Cml catalyzed by MarH with different acyl-CoAs. (i) Cml with boiled MarH; (ii) 5 incubated with MarH for 15 min, and (iii) 5, (iv) 6, (v) 7 as donors incubated with MarH for 2 h. (C) Proposed acylation of Cml catalyzed by MarH.

Figure 2. (A) Catalytic efficiency of MarH and its mutants. (B) Proposed mechanism of MarH-catalyzed Cml acetylation.

transition state (Figure S2B).19 These results augured that MarH could not directly transfer the acetyl group to the 1hydroxy group of Cml and also indicated that the diacetylated product 4 was derived from the acetylation of 3, not 2 (Figure 1C). To confirm this hypothesis, we conducted two parallel reactions with either purified 2, or the mixture of 2 and 3 at equilibrium composition. In the reaction with 2, 4 was barely formed until the transformation of 2 to 3 reached the equilibrium at 20 min. After this time point, 4 steadily increased, along with the gradual decrease of 2, while 3 remained unchanged (Figure S3A). In contrast, in the reaction with the mixture of 2 and 3, 4 was produced from the beginning of the reaction and steadily increased along with the decrease of 3, but 2 almost remained unchanged until the reaction proceeded for 30 min (Figure S3B). These results established regiospecificity of MarH for the 3-O-acetylation of Cml. Overall, the acetylation of Cml catalyzed by MarH is similar to that of CATs (Figure 1C).20 CATs catalyze the acetylation of Cml using a conserved histidine residue as the catalytic base to activate the acetylacceptor. MarH belongs to the cupin superfamily without any homology to CATs. Sequence alignment of MarH and several cupin superfamily proteins revealed the conserved amino acid

residues H62, H64, and E68 of the motif 1 (HxHxxxE) as well as P102, G104, and H107 of the motif 2 (PxGxxH) that are characteristics of the cupin superfamily proteins (Figure S4).21 Five additional amino acid residues F31, F38, P75, C120, and G122 were shared by MarH and canonical CATs (Figure S4). In order to locate the key residues for acyl transfer activity of MarH, site-directed mutagenesis of these amino acid residues was performed, and the resulting mutant proteins of MarH were overexpressed and purified (Figure 2A and S5). All mutant proteins were confirmed to retain the overall protein structure of MarH by circular dichroism (CD) spectra (Figure S6). The enzymatic efficiencies of MarH and the mutants were determined (SI). The activities of the F31A and F38A mutants increased in about 50%. It was speculated that the replacement of phenylalanine by alanine provided more space for Cml binding. The mutant H64A exhibited significantly reduced CAT activity, whereas other mutants retained comparative activities to the wild type MarH (Figure 2A). Taken together, H64 might act as a catalytic base in the acetylation of Cml catalyzed by MarH, which is similar to CATs and other acetyltransferase.22 The 3-hydroxy group of Cml might be deprotonated by the imidazole of the H64 residue. Nucleophilic 789

DOI: 10.1021/acscatal.5b02198 ACS Catal. 2016, 6, 788−792

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ACS Catalysis attack at the carbonyl carbon of acetyl-CoA completes the acetylation of Cml (Figure 2B). To confirm this speculation, CAT activity of MarH was tested at different pH values (SI). MarH showed relatively higher CAT activity under alkaline conditions that are beneficial for deprotonation (Figure S7). In addition, four acidic residues (D36, E47, D87, and E92), which might deprotonate H64, were also mutated to Ala, but the resultant mutation had no effect on CAT activity (Figure 2A). Intriguingly, H107 from motif 2 had been confirmed as a catalytic base for the epimerase activity,18 whereas H64 from motif 1 was demonstrated now to act as the catalytic base for the CAT activity. Structural studies to reveal why MarH can catalyze both the epimerization and acylation are in progress. In our previous work, three homologues of MarH including StnK3 (AFW04574), ACPL_6197 (AEV87082), and Cwoe_4835 (ADB53248) could catalyze the same epimerization with similar efficiency as MarH.18 All three proteins were tested whether they could also catalyze the acetylation of Cml. Both the monoacetylated and diacetylated products of Cml (2, 3, and 4) were detected in the typical in vitro assays (Figure S8). Furthermore, the steady-state kinetic parameters for these four proteins were acquired with varying Ac-CoA concentrations (SI and Figure S9) and summarized in Table 1. MarH and StnK3 showed similar catalytic efficiency to the canonical CATs.20,23

Acyltransferases are widely distributed in nature. They not only play important roles in life processes24 but also participate in the biosynthesis of numerous secondary metabolites.25 Moreover, several acyltransferases have been successfully applied for the synthesis of pharmaceuticals.26 For medicinal applications, acylated derivatives often have improved biological activities, stabilities, or availabilities, compared to the original compounds.27 Because no acyltransferase has ever been reported from the cupin superfamily, we were curious about whether MarH can recognize other acyl donor substrates. Butyryl-CoA (6), isobutyryl-CoA (7), and six other acyl CoAs (8−13) were tested as putative acyl donors in typical assays (Figure S10). HPLC and HR-MS analysis showed that only monobutyrylated and monoisobutyrylated products of Cml (14−17) were formed in the reactions with 6 and 7 as acyl donors (Figure 1B and Table S2). The structure of 14 was confirmed by NMR analysis (Figure S21−22). The position of acylation is easily deducible from the chemical shift of the 3-H that was found at δH = 3.74−3.60 ppm for Cml28 compared to δH = 4.32−4.14 ppm for the corresponding butyrylated compound (Table S4). 16 was identified only by HR-MS due to limited amounts of substrate 7 (Table S2 and Figure S14). Comparative kinetic parameters were obtained for 6 as a donor substrate (Km = 185.4 ± 29.50 μM and kcat = 1.56 ± 0.08 min−1). It is notable that no dibutyrylated or diisobutyrylated Cml could be detected even by increasing the enzyme concentration and extending the reaction time, probably due to the increased steric hindrance of the butyryl and isobutyryl groups. No product was observable in the reactions with other putative acyl donors, implying that the variability of acyl donors recognized by MarH is limited. However, compared to canonical CATs,23 MarH still presents a relatively flexible substrate specificity for acyl donors.

Table 1. Kinetic Parameters for MarH and Its Homologues enzyme MarH StnK3 ACPL_6197 Cwoe_4835

Km (μM) 88.39 88.72 72.91 104.9

± ± ± ±

5.99 2.68 8.81 20.40

kcat (min−1)

kcat/Km (mM−1 min−1)

± ± ± ±

15.12 22.54 9.19 4.39

1.26 2.00 0.67 0.46

0.04 0.01 0.02 0.02

Figure 3. Acceptor substrate specificity of MarH. (A) Structures of the putative acceptors; (B) HPLC profiles of acetylation catalyzed by MarH with Ac-CoA and putative acceptors. (i) 18 and (iii) 19 incubated with boiled MarH for 3 h; (ii) 18 and (iv) 19 incubated with MarH for 3 h. (C) Proposed acetylation of 18 catalyzed by MarH. 790

DOI: 10.1021/acscatal.5b02198 ACS Catal. 2016, 6, 788−792

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ACS Catalysis To gain insights into the acceptor substrate specificity, chemo- and stereoselectivity of MarH, compounds with similar structures as Cml (18−21) were tested as putative acetyl acceptors in the in vitro assays (Figure 3A). HPLC and HR-MS analysis revealed that only 18 was acetylated by MarH, generating four acetylated products (Figure 3B and Table S3). To elucidate the structures of these products, 23, 24, and 25 were purified from a biotransformation of 18 using E. coli BL21(DE3) containing pET28a-marH. NMR analysis (SI) revealed that 23 and 24 were indeed diacetylated products while 25 was the triacetylated product of 18 (Figure S23−S37). As before, the acylation position was assigned on the basis of chemical shift variations of the adjacent hydrogen atoms and furthermore confirmed by HMBC data. Notably, the amino group of all three products was acetylated. However, only trace amounts of 22 could be obtained that were insufficient for NMR analysis. Because the mono-O-acetylated products of Cml were always observed as an interconvertible mixture and no further peaks with the same HR-MS as 22 were found, we concluded that 22 might be the N-acetylated product of 18. The low amounts of 22 accumulated in the biotransformation of 18 by MarH implicated that MarH first catalyzed the acetylation of the amino group of 18, further initiating the 3-Oacetylation of 18 that proceeded with a faster reaction rate. To verify this assumption, we carried out time-dependent enzymatic assays of MarH with 18 and Ac-CoA as substrates (Figure S11). This time-course analysis not only confirmed this hypothesis but also showed that 23 could spontaneously react to 24 that was further acetylated to 25 (Figure 3C and S12). MarH could catalyze the acetyl transfer to 18, but not to 19, exhibiting the stringent (1R,2R)-selectivity. Because 20 and 21 could not be acetylated by MarH, we speculated that the nitro group is essential for substrate binding to MarH. Taken together, MarH was proven to be a robust enzyme that not only functions as an epimerase18 but also acts as an O- and Nacyltransferase. Finally, to test whether marH could function as a Cml resistance gene in vivo, the anti-Cml bioassay was carried out using E. coli BL21(DE3) strains containing the expression plasmid pET28a-marH and the control plasmid pET28a as indicator strains, respectively. The strain E. coli BL21(DE3)/ pET28a cannot grow on LB-agar medium containing Cml while the strain E. coli BL21(DE3)/pET28a-marH showed an anti-Cml effect (Figure S13). These findings indicated that MarH can be used not only as a robust acyltransferase but also as a Cml resistance gene in vivo. In summary, we have discovered a group of small cupin proteins (129−132 aa residues), which can catalyze not only epimerization of β-methylindolepyruvate but also esterification and amide bond formation of Cml by acting as a dual acyltransferase. Studies of the reaction kinetics indicated that these small cupin proteins had comparative catalytic efficiency to canonical CATs. By systematic point mutation study, H64 was discovered to be the key active site and a similar catalytic model was proposed to that of CATs. Overall, MarH showed multifunctional properties, flexible substrate specificity, and strict regio- and enantioselectivity, providing a potential to be explored as an efficient biocatalyst used for organic synthesis.

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Experimental details, NMR, Q-TOF, and other supplemental data (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ‡

These authors contributed equally (M.H. and H.Y.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Wen Liu at Shanghai Institute of Organic Chemistry CAS for gifting acyl CoAs. This work was financially supported by the National Natural Science Foundation of China (31425001 and 21372154 for S.L.; 31121064 for Z.D.) and the grants from MOE of China and the Leopoldina Fellowship Program (German National Academy of Sciences Leopoldina, LPDS 2013-12 for N.L.B.).

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.5b02198. 791

DOI: 10.1021/acscatal.5b02198 ACS Catal. 2016, 6, 788−792

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DOI: 10.1021/acscatal.5b02198 ACS Catal. 2016, 6, 788−792