Background: Polysaccharide monooxygenases (PMOs) of the auxiliary activity 9 (AA9) family have been reported to oxidize C1, C4, and C6 positions in cellulose. However, currently no direct evidence exists that PMOs oxidize C6 posi- tions in cellulose, and molecular mechanism of C1, C4 and C6 oxidation is unclear. Results: In this study, a PMO gene (Ctpmo1) belonging to AA9 was isolated from Chaetomium thermophilum and suc- cessfully expressed and correctly processed in Pichia pastoris. A simple and effective chemical method of using Br to oxidize CtPMO1 reaction products was developed to directly identify C4- and C6-oxidized products by matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF–MS). The PMO (CtPMO1) cleaves phosphoric acid-swollen cellulose (PASC) and celloheptaose, resulting in the formation of oxidized and nonoxidized oligosaccharides. Product identification shows that the enzyme can oxidize C1, C4, and C6 in PASC and cello-oligosac- charides. Mutagenesis of the aromatic residues Tyr27, His64, His157 and residue Tyr206 on the flat surface of CtPMO1 was carried out using site-directed mutagenesis to form the mutated enzymes Y27A, H64A, H157A, and Y206A. It was demonstrated that Y27A retained complete activity of C1, C4, and C6 oxidation on cellulose; Y206A retained partial activity of C1 and C4 oxidation but completely lost activity of C6 oxidation on cellulose; H64A almost completely lost activity of C1, C4, and C6 oxidation on cellulose; and H157A completely lost activity of C1, C4, and C6 oxidation on cellulose. Conclusions: This finding provides direct and molecular evidence for C1, C4, especially C6 oxidation by lytic polysac- charide monooxygenase. CtPMO1 oxidizes not only C1 and C4 but also C6 positions in cellulose. The aromatic acid residues His64, His157 and residue Tyr206 on CtPMO1 flat surface are involved in activity of C1, C4, C6 oxidation. Keywords: Chaetomium thermophilum, Auxiliary activity family 9 (AA9), Polysaccharide monooxygenase (PMO), Regioselectivity of oxidation, C1, C4 and C6 oxidation Background production . Recently, a new class of cellulose-degrad- 2+ Cellulose is one of the most abundant renewable carbo- ing enzymes, called Cu -dependent lytic polysaccharide hydrates on earth. Enzymatic degradation of cellulose monooxygenases (PMOs) , have been discovered and to glucose has great potential for biofuel production are attracting increasing interest because their oxidative . This enzymatic degradation is thought to be accom - degradation of cellulose dramatically boosts cellulase plished by the synergistic action of three classes of cellu- activity in cellulose hydrolysis [5–8]. lases: endocellulases, exocellulases, and beta-glucosidases Based on their amino acid sequence similarities, PMO . However, the low degradation efficiency and the high enzymes are classified into four families: auxiliary activ - cost of cellulases are major barriers to economical biofuel ity 9 (AA9), auxiliary activity 10 (AA10), auxiliary activ- ity 11 (AA11), and auxiliary activity 13 (AA13) [4, 9, 10]. With a plethora of biochemical, structural, functional, *Correspondence: firstname.lastname@example.org and regioselective data available, PMOs from AA9 have Department of Mycology, Shandong Agricultural University, Taian 271018, Shandong, China © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. Biotechnol Biofuels (2018) 11:155 Page 2 of 16 been studied intensively in fungi [7, 8, 11–18]. From a reaction (PCR) with a pair of specific primers from the structural perspective, the 3-D structure of AA9 PMOs, CGMCC3.17990 strain of C. thermophilum from China to perform cleavage, displays a beta-sheet fold with a flat (Additional file 1: Table S1). The KC441882 gene was substrate-binding surface that differs from traditional designated as Ctpmo1, encoding a putative AA9 PMO cellulases in that it does not show a substrate-binding protein, CtPMO1. The CtPMO1 protein was predicted cleft (groove, crevice, or tunnel). The flat surface of an to be a secreted enzyme with a 17-amino acid potential AA9 PMO binds cellulose molecules and contains at its signal peptide. The mature CtPMO1 protein is composed center an absolutely conserved N-terminal histidine resi- of 229 amino acids with a calculated molecular weight of due where a copper ion is confirmed to bind tightly [5, 24.63 kDa. Like most AA9 proteins, CtPMO1 has only a 7, 14, 19]. With respect to the oxidation position of sub- catalytic domain and no additional modules . strate molecules, AA9 PMO enzymes have demonstrated To obtain the CtPMO1 protein with a native striking differences in regioselectivities, most possibly N-terminus, we used the plasmid pPICZαA for because of their multigenicity [4, 11]. The enzymes oxi - Ctpmo1 expression in P. pastoris. After induc- dize the C1 carbon atom of glucose, but may also oxidize tion with methanol, we purified the expressed 2+ C4 or even C6. Through an elimination reaction, the C1 Cu -loaded CtPMO1 using nickel affinity chroma - 2+ and C4 oxidation results in two direct cleavage sites of the tography from the Cu -containing culture filtrate of glucosidic bond. The C1 oxidation leads to the formation P. pastoris transformed with the recombinant plasmid of sugar lactone, which is spontaneously hydrolyzed into pPICZαA/Ctpmo1 (Additional file 1: Figure S1). Using aldonic acid, and the C4 and C6 oxidations lead to the SDS-PAGE, we estimated the molecular weight of the formation of C4-ketoaldose [8, 11, 12, 16, 17, 20, 21] and purified recombinant CtPMO1, which contains a 6× His C6-hexodialdose [7, 15], respectively. However, no direct tag and a myc tag (2.68 kDa) at the C-terminal, to be evidence demonstrates the presence of C6-hexodialdose approximately 27.5 kDa. Subtracting 2.68 kDa brought in PMO’s reaction products [12, 17]. Although PMO this value very close to the 24.63 kDa, we calculated C6 oxidation has been suggested based only on mass from the deduced amino acid sequence of CtPMO1. measurements [7, 15], mass measurements alone are We identified the N-terminal amino acid sequence of not enough to confirm that PMOs can oxidize C6 posi - CtPMO1 using LC-MS/MS to be HAIFQK (Additional tions of cellulose, because C6-hexodialdose has the same file 1: Figure S2), indicating that CtPMO1 was correctly molecular weight as C4-ketoaldose . Because PMO processed in P. pastoris. As in the case of other PMOs C6 oxidation has not been rigorously demonstrated [12, expressed in P. pastoris [16, 25], the N-terminal His res- 17], the described three groups of AA9 PMOs (PMO1 for idue in CtPMO1 is not methylated. C1 oxidation, PMO2 for C4 oxidation, and PMO3 for C1 and C4 oxidation) are not involved in C6 oxidation . Identification of CtPMO1 soluble reaction products Currently, PMO C6 oxidation has remained elusive and To identify products of CtPMO1, we performed solu- considerable academic debate is being conducted regard- ble product assay on phosphoric acid-swollen cellulose ing PMO C6 oxidation [11, 12, 17, 21, 22]. (PASC) using TLC and MALDI-TOF–MS analysis. TLC Chaetomium thermophilum is a thermophilic fungus analysis showed that treatment of PASC with CtPMO1 and can grow in temperatures up to 60 °C. The thermo - mainly produced cello-oligosaccharides with a degree of philic fungus has been suggested as a model organism polymerization (DP) from DP to DP (Fig. 1). MALDI- 2 6 for biochemical and prospective structural analyses of TOF–MS analysis showed that treatment of PASC with eukaryotic macromolecular complexes and biotechno- CtPMO1 produced a series of molecular ions with m/z logical applications of thermostable eukaryotic proteins corresponding to cello-oligosaccharide products with a [23, 24]. C. thermophilum genome analysis reveals 19 DP from DP to DP (Fig. 2). Importantly, the molecular 3 6 genes encoding putative AA9 PMOs (http://www.funga ions corresponding to various oxidized oligosaccharides, lgeno mics.cn). In the present paper, we provide direct C1-oxidized oligosaccharides (aldonic acid, m/z + 16) evidence that an AA9 PMO from C. thermophilum dis- and C4- or C6-oxidized oligosaccharides (C4-ketoaldose plays oxidation at C1, C4, and C6 positions of cellulose, or C6-hexodialdose, m/z − 2), were observed, indicat- especially at C6 positions of cellulose. ing the nature of oxidative enzymes. Additionally, minor double C4 and C6 oxidized oligosaccharides (m/z − 4) Results and double C1 and C4 or C6 oxidized oligosaccharides CtPMO1 expression and purification (m/z + 14) were observed. MS/MS fragmentation of the We amplified a gene (KC441882) with only one amino highest peak with m/z value of 525 from MALDI-TOF– acid difference from the Chath2p7_007187 gene MS analysis, corresponding to the C6 or C4-oxidized of C. thermophilum genome by polymerase chain products (DP -2), showed the presence of nonoxidized 3 Chen et al. Biotechnol Biofuels (2018) 11:155 Page 3 of 16 (C6-hexodialdose) in CtPMO1 soluble reaction prod- ucts. As expected, H NMR spectrum of CtPMO1 soluble reaction products displayed an aldehyde pro- ton signal at δ 8.39 (Additional file 1: Figure S5). The anomeric resonance at δ 8.39 is close to the anomeric resonances at δ 9.19 and 9.50 that are assigned to the aldehyde proton of two C6-oxidized galactose prod- ucts . To confirm CtPMO1 C4 and C6 oxidation, we per - formed another chemical method, using Br to oxidize CtPMO1 soluble reaction products for MALDI-TOF– MS analysis. Based on the chemical method, if CtPMO1 products had C1-oxidized oligosaccharides (m/z + 16), they would not be oxidized by Br ; if CtPMO1 prod- ucts had C4-oxidized oligosaccharides (C4-ketoaldose, Fig. 1 Analysis of CtPMO1 soluble reaction products with PASC as m/z − 2), they would be oxidized by Br to form C4- substrate using TLC. Soluble reaction products upon incubation and C1-oxidized oligosaccharides (4-keto-aldonic acid, of 0.5% PASC with CtPMO1 in 10 mM HAc-NH Ac (pH 5.0) and m/z + 14); if CtPMO1 products had C6-oxidized oli- 1 mM ascorbate at 50 °C for 0, 12, 24, 36, and 48 h. M, standard cellulo-oligosaccharides (G1–G7) gosaccharides (C6-hexodialdose, m/z − 2), they would be oxidized by Br to form C6- and C1-oxidized cello- oligosaccharides (m/z + 30); and if CtPMO1 products fragmentation ions and C6 or C4-oxidized fragmenta- had C4- and C6-oxidized oligosaccharides (m/z − 4), tion ions (Additional file 1: Figure S3, S4, Table S2). In they would be oxidized by Br to form C4-, C6- and particular, we observed that the potential fragmentation C1-oxidized cello-oligosaccharides (m/z + 28) (Fig . 3a). ions oxidized only at C6, indicating that the C6 positions As expected, we observed the molecular ions corre- could be modified. sponding to various oxidized oligosaccharides, C4- Because NMR has been applied to identify galac- and C1-oxidized oligosaccharides (m/z + 14), C6- and tose oxidation at C6 positions , it was performed C1-oxidized oligosaccharides (m/z + 30), and C6-, to confirm C6-oxidized cellulo-oligosaccharides C4-, and C1-oxidized oligosaccharides (m/z + 28) in Fig. 2 Identification of CtPMO1 soluble reaction products with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h 4 Chen et al. Biotechnol Biofuels (2018) 11:155 Page 4 of 16 Fig. 3 Identification of CtPMO1 soluble reaction products oxidized by Br with PASC as substrate using MALDI-TOF–MS. Reaction pathway for oxidation of cellulose by CtPMO1 followed Br oxidation (a) and reaction products oxidized by Br (b). C1-oxidized oligosaccharides (m/z + 16), 2 2 C4- and C1-oxidized oligosaccharides (m/z + 14), C1- and C6-oxidized oligosaccharides (m/z + 30), and C1-, C6- and C4-oxidized oligosaccharides (m/z + 28) Chen et al. Biotechnol Biofuels (2018) 11:155 Page 5 of 16 CtPMO1 C4‑ and C1‑oxidized products are also present CtPMO1 soluble reaction products oxidized by Br in its insoluble reaction products using MALDI-TOF–MS analysis (Fig. 3b). The pro - To confirm whether CtPMO1 products also exist in its portion of C6-oxidized oligosaccharides (m/z + 30) is insoluble reaction products (residual PASC), CtPMO1 about 1/10–1/5 of C4-oxidized oligosaccharides (m/z insoluble reaction products hydrolyzed by endo- + 14), and the proportion of C6-oxidized oligosaccha- 1,4-beta-glucanase followed by Br oxidation were iden- rides (m/z + 30) and C4- and C6-oxidized oligosac- tified using MALDI-TOF–MS analysis (Fig. 4). Just as charides (m/z + 28) is about 1/6–1/3 of C4-oxidized expected, nonoxidized cello-oligosaccharides (m/z + 0), oligosaccharides (m/z + 14), indicating that C4-oxi- C1-oxidized oligosaccharides (m/z + 16) and C4- or dized oligosaccharides is predominant in CtPMO1 sol- C6-oxidized oligosaccharides (m/z − 2) were released uble reaction products. Fig. 4 CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase followed by Br oxidation with PASC as substrate. Insoluble reaction products (residual PASC) upon incubation of 0.5% PASC with CtPMO1 in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase using TLC analysis (a) and MALDI-TOF–MS (b). CtPMO1 insoluble reaction products hydrolyzed by endo-1,4-beta-glucanase followed by Br oxidation for 30 min (c) and for 60 min (d) using MALDI-TOF–MS analysis. C1-oxidized oligosaccharides (m/z + 16), C4- or C6-oxidized oligosaccharides (m/z − 2), lactones (m/z -4) of C1- (m/z + 16) and C4- or C6-oxidized oligosaccharides (m/z − 2), C4- and C1-oxidized oligosaccharides (m/z + 14), C6- and C1-oxidized oligosaccharides (m/z + 30), and C6-, C1- and C4-oxidized oligosaccharides (m/z + 28). M, standard cellulo-oligosaccharides (G1–G7). CK, samples upon incubation of insoluble reaction products (residual PASC) in 10 mM HAc-NH Ac (pH 5.0) at 50 °C for 10 min with inactive endo-1,4-beta-glucanase treated at 100 °C for 30 min 4 Chen et al. Biotechnol Biofuels (2018) 11:155 Page 6 of 16 from CtPMO1 insoluble reaction products hydrolyzed analysis (Fig. 5b), indicating that CtPMO1 can oxidize by endo-1,4-beta-glucanase (Fig. 4a, b). Unexpectedly, C4, C1 and C6 positions of soluble oligosaccharides. oxidized oligosaccharides with DP (m/z − 4) were obvi- ously observed in CtPMO1 insoluble reaction prod- Structural model of CtPMO1 ucts hydrolyzed by endo-1,4-beta-glucanase. After Br To understand the molecular basis of CtPMO1 C1, oxidation, we observed the main molecular ions cor- C4 and C6 oxidation, the 3-D structure of its catalytic responding to C1-oxidized oligosaccharides (m/z + 16) domain was predicted on the basis of known 3-D struc- and C4- and C1-oxidized oligosaccharides (m/z + 14) tures of PMO proteins using homology modeling [5, and the minor molecular ions corresponding to C6- and 7, 14, 19, 27]. CtPMO1 shares a high identity of 64.32% C1-oxidized oligosaccharides with DP (m/z + 30), but with a PMO (NcLPMO9C) of Neurospora crassa (Addi- failed to observe molecular ions corresponding to C6- tional file 1: Figure S6), whose 3-D structure (PDB id and C1-oxidized oligosaccharides with DP (m/z + 30) 4D7U) has been reported . Therefore, the homology and C6-, C4-, and C1-oxidized oligosaccharides (m/z model of CtPMO1 was obtained using NcLPMO9C as a + 28), in CtPMO1 insoluble reaction products hydro- template. The homology model shows that three highly lyzed by endo-1,4-beta-glucanase followed by Br oxida- conserved amino acid residues, His1, His83 and Tyr168, tion for 30 min (Fig. 4c) and 60 min (Fig. 4d). The absence are clustered at the flat surface near the N-terminus of the molecular ions corresponding to C6-, C4-, and of CtPMO1, and four aromatic residues Tyr27, His64, C1-oxidized oligosaccharides (m/z + 28) shows that the His157 and Tyr206 (two conserved residues His157 and oxidized oligosaccharides with DP (m/z − 4) in CtPMO1 Tyr206, and two varied residues Tyr27, His64) are pre- insoluble reaction products hydrolyzed by endo-1,4-beta- sent on the flat surface of CtPMO1 (Additional file 1: Fig- glucanase are lactones (m/z − 2) of C4 or C6-oxidized ure S7). The N-terminal amino group (2.3 Å), the Nδ of (m/z − 2) aldonic acid (m/z + 16) rather than C6- and His1 (2.2 Å), and the Nε of His83 (2.0 Å) form a copper C4-oxidized oligosaccharides (m/z − 4). These data indi - ion-binding site (a histidine-brace). The highly conserved cate that C6-oxidized products are minor in CtPMO1 residue Tyr168 is buried and lies in the protein-facing insoluble reaction products. It should be pointed out that axial position with a distance of 3.0 Å from the copper the molecular ion peaks (m/z − 2) dramatically increased, to the oxygen atom of the Tyr168 side chain. The three but the molecular ion peaks (m/z + 14 and + 30) did not highly conserved residues His1, His83, and Tyr168 coor- obviously increase, in CtPMO1 insoluble reaction prod- dinate the copper ion essential for catalysis [11, 27]. The ucts hydrolyzed by endo-1,4-beta-glucanase followed by role in catalytic activity of the aromatic residues Tyr27, Br oxidation. The most possible reason for this is that His64, His157, and Tyr206 on the flat surface of CtPMO1 the molecular ion peaks (m/z − 2) correspond to lactones remains unclear (Additional file 1: Figure S8). (m/z − 2) of C1-oxidized oligosaccharides (aldonic acid, m/z + 16), which are largely produced from nonoxidized Role of the aromatic residues Tyr27, His64, His157 oligosaccharides by Br oxidation and easily converted to and Tyr206 on the flat surface of CtPMO1 their lactones (m/z − 2) in solution. To determine whether the aromatic residues Tyr27, His64, His157 and Tyr206 on the flat surface of CtPMO1 CtPMO1 can C1‑, C4‑, and C6‑oxidize soluble celloheptaose are involved in catalytic activity of C1, C4 and C6 oxi- To confirm C1, C4, and C6 oxidation of CtPMO1 on dation, mutation of the four residues of the wild-type soluble cello-oligosaccharides, we used celloheptaose as CtPMO1 enzyme (WT) was carried out using site- substrates of CtPMO1 to generate oxidized cellulo-oligo- directed mutagenesis to form four mutated enzymes: saccharides. MALDI-TOF–MS/MS analysis confirmed Y27A, H64A, H157A and Y206A. TLC analysis of solu- the presence of C1-oxidized oligosaccharides (aldonic ble reaction products showed that cello-oligosaccharides acid, m/z + 16) and C6- or C4-oxidized oligosaccha- were observed in Y27A, a minor amount of cello-oligo- rides (C6-hexodialdose or C4-ketoaldose, m/z − 2) upon saccharides were observed in Y206A, and no cello-oligo- incubating CtPMO1 with celloheptaose (Fig. 5a). Like saccharides were observed in H64A and H157A (Fig. 6). CtPMO1 soluble reaction products oxidized by Br with MALDI-TOF–MS analysis showed that C1-oxidized PASC as substrate, we also observed the molecular ions (m/z + 16) and C4- or C6-oxidized (m/z − 2) cello-oligo- corresponding to C4- and C1-oxidized oligosaccharides saccharides were observed in Y27A and Y206A, a minor (m/z + 14), C6- and C1-oxidized oligosaccharides (m/z amount of C1-oxidized (m/z + 16) and C4- or C6-oxi- + 30), and C6-, C4- and C1-oxidized oligosaccharides dized (m/z − 2) cello-oligosaccharides were observed (m/z + 28), in CtPMO1 reaction products oxidized by Br in H64A, and no oxidized cello-oligosaccharides were with celloheptaose as substrate using MALDI-TOF–MS observed in H157A (Fig. 7). Chen et al. Biotechnol Biofuels (2018) 11:155 Page 7 of 16 Fig. 5 Identification of CtPMO1 reaction products oxidized by Br with celloheptaose as substrate using MALDI-TOF–MS. Reaction products upon incubation of 0.1% celloheptaose with CtPMO1 in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. Reaction products (a) and reaction products oxidized by Br (b). C1-oxidized oligosaccharides (m/z + 16), C4- or C6-oxidized oligosaccharides (m/z − 2), C4- and C1-oxidized oligosaccharides (m/z + 14), C6- and C1-oxidized oligosaccharides (m/z + 30), and C1-, C6- and C4-oxidized oligosaccharides (m/z + 28) To confirm the involvement of the residues Tyr27, (m/z + 28) were observed in H64A, a minor amount of His64, His157 and Tyr206 of CtPMO1 in C4 and C6 oxi- C4- and C1-oxidized oligosaccharides (m/z + 14) but no dation, soluble reaction products of the mutated enzymes C6- and C1-oxidized oligosaccharides (m/z + 30) and were oxidized by Br for MALDI-TOF–MS analysis as C1-, C4- and C6-oxidized oligosaccharides (m/z + 28) the WT enzyme. As expected, C4- and C1-oxidized oli- were observed in Y206A, and no oxidized oligosaccha- gosaccharides (m/z + 14), C6- and C1-oxidized oligo- rides were observed in H157A (Fig. 8). Similar analy- saccharides (m/z + 30), and C1-, C4- and C6-oxidized sis of insoluble reaction products showed that C4- and oligosaccharides (m/z + 28) were observed in Y27A, a C1-oxidized oligosaccharides (m/z + 14) and C6- and minor amount of C4- and C1-oxidized oligosaccharides C1-oxidized oligosaccharides (m/z + 30) were observed (m/z + 14), C6- and C1-oxidized oligosaccharides (m/z in Y27A, a minor amount of C4- and C1-oxidized oligo- + 30), and C1-, C4- and C6-oxidized oligosaccharides saccharides (m/z + 14) were observed in H64A, and no Chen et al. Biotechnol Biofuels (2018) 11:155 Page 8 of 16 reaction products for identifying C4- and C6-oxidized products. The method has three potential advantages. First, because there is no interference by metal ions (N a and Ag), Br oxidation of CtPMO1 products allows us to directly identify C4- and C6-oxidized products using MALDI-TOF–MS analysis, but there is interference of + + metal ions (N a and Ag ) in CtPMO1 products oxidized by I . Second, residual Br in CtPMO1 products oxidized 2 2 by Br is very easily removed under a stream of nitrogen at 40 °C, unlike the removal of I which requires add- ing excess Ag CO in CtPMO1 products oxidized by I . 2 3 2 Third, Br oxidation of CtPMO1 reaction products pro- vided acidic conditions that can prevent the generation of unsaturated oligosaccharides, unlike oxidized by I under alkali conditions. CtPMO1 oxidizes PASC to produce C1-, C4-, and C6-oxidized products in its soluble reaction products Fig. 6 Identification of the mutated CtPMO1 soluble reaction and C4- and C1-oxidized products in its insoluble reac- products with PASC as substrate using TLC. Soluble reaction tion products, but C6-oxidized products are minor in its products upon incubation of 0.5% PASC with the mutated CtPMO1 insoluble reaction products. One possible explanation enzymes (Y27A, H64A, H157A and Y206A) in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. M, standard for this is that C6-oxidized oligosaccharides in CtPMO1 cellulo-oligosaccharides (G1–G7). CK, samples upon incubation of soluble reaction products may be mainly produced by 0.5% PASC in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at C6 oxidation of C4- and C1-cleaved soluble oligosaccha- 50 °C for 48 h with inactive CtPMO1 treated at 100 °C for 30 min rides (oxidized and nonoxidized oligosaccharides). This explanation is supported by the evidence that CtPMO1 C6-oxidizes soluble celloheptaose to produce C6-oxi- dized oligosaccharides and that LsAA9A and CvAA9A C4- and C1-oxidized oligosaccharides (m/z + 14) and C4-oxidize small oligosaccharides, whereas they C4- and C6- and C1-oxidized oligosaccharides (m/z + 30) were C1-oxidize polysaccharides . Conformational flex - observed in H157A and Y206A (Fig. 9). ibility of soluble cello-oligosaccharides possibly causes Together, these data indicate that the mutated enzyme the changes in oxidation type (C1, C4, and C6) of a Y27A retained complete activity of C1, C4, and C6 oxi- PMO enzyme. It should be pointed out that three PMOs dation on cellulose; the mutated enzyme Y206A retained (TaGH61A, PaGH61B and CtPMO1) identified to oxidize partial activity of C1 and C4 oxidation but completely lost C6 position of cellulose ([7, 15], this study) have a long activity of C6 oxidation on cellulose; the mutated enzyme reaction time (TaGH61A for 22 h, PaGH61B for 48 h and H64A almost completely lost activity of C1, C4, and C6 CtPMO1 for 48 h) to produce C6-oxidized oligosaccha- oxidation on cellulose; and the mutated enzyme H157A rides. The long reaction time also hints that C6-oxidized completely lost activity of C1, C4, and C6 oxidation on oligosaccharides may be produced from C4- and/or cellulose (Table 1), suggesting that the residue His157 in C1-oxidize polysaccharides by PMO C6 oxidation. CtPMO1 is required for activity of C1, C4, and C6 oxi- It has been suggested that aromatic residues on the dation on cellulose; the residue His64 in CtPMO1 plays PMO protein flat surface are involved in substrate bind - a key role in retaining activity of C1, C4, and C6 oxida- ing [14, 27, 28]. In this study, mutation of the residue tion on cellulose; the residue Tyr206 in CtPMO1 plays a His157 in CtPMO1 results in complete loss of activ- partial role in retaining activity of C1 and C4 oxidation ity of C1, C4, and C6 oxidation on cellulose, maybe but a key role in retaining activity of C6 oxidation on cel- because the residue His157 is adjacent to the copper lulose; and the residue Tyr27 in CtPMO1 may not play ion-binding site of CtPMO1. Interestingly, mutation of an important role in retaining activity of C1, C4, and C6 the residue His64 in CtPMO1 results in almost com- oxidation on cellulose. plete loss of activity of C1, C4, and C6 oxidation on cel- lulose. The residue His64 lies in a sequence insertion (L3 Discussion loop) that seems unique for C4-oxidizing PMOs . Cello-oligosaccharides containing a C6 aldehyde can be It has been reported that an additional metal-binding oxidized by I and Br to the corresponding uronic acids 2 2 site (copper or zinc ion) is coordinated by the residue . In this study, we developed a simple and effec - His64 in NcLPMO9C from N. crass . Recent studies tive chemical method, using Br to oxidize CtPMO1 2 Chen et al. Biotechnol Biofuels (2018) 11:155 Page 9 of 16 Fig. 7 Identification of the mutated CtPMO1 soluble reaction products with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes Y27A (a), H64A (b), H157A (c) and Y206A (d) in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides (aldonic acid, m/z + 16) and C4- or C6-oxidized oligosaccharides (C4-ketoaldose or C6-hexodialdose, m/z − 2) of LsAA9A-substrate interaction show that a hydrogen role in PMO–substrate interaction. Mutation of the resi- bond is present between the residue His66 in LsAA9A due Tyr206 in CtPMO1 results in partial loss of activity (His64 in CtPMO1 and NcLPMO9C) and O3 at substrate of C1 and C4 oxidation and complete loss of C6 oxida- + 2 . These data suggest that the residue His (His64 tion on cellulose, may be because the residue Tyr206 in CtPMO1 and NcLPMO9C, His66 in LsAA9A) plays a is far away from the copper ion-binding site (at subsite (See figure on next page.) Fig. 8 Identification of the mutated CtPMO1 soluble reaction products oxidized by Br with PASC as substrate using MALDI-TOF–MS. Soluble reaction products upon incubation of 0.5% PASC with the mutated CtPMO1 enzymes Y27A (a), H64A (b), H157A (c) and Y206A (d) in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides (m/z + 16), C4-oxidized oligosaccharides (m/z + 14), C6-oxidized oligosaccharides (m/z + 30), and C6- and C4-oxidized oligosaccharides (m/z + 28) Chen et al. Biotechnol Biofuels (2018) 11:155 Page 10 of 16 Chen et al. Biotechnol Biofuels (2018) 11:155 Page 11 of 16 Fig. 9 Mutated CtPMO1 insoluble reaction products oxidized by Br with PASC as substrate using MALDI-TOF–MS. Insoluble reaction products (residual PASC) upon incubation of 0.5% PASC with mutated CtPMO1 Y27A (a), H64A (b), H157A (c) and Y206A (d) in 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate at 50 °C for 48 h. C1-oxidized oligosaccharides (m/z + 16), C4-oxidized oligosaccharides (m/z + 14), and C6-oxidized oligosaccharides (m/z + 30) Table 1 Composition of CtPMO1 and its mutants’ reaction products with PASC as substrate Enzyme Soluble reaction products Insoluble reaction products C1 C4 C6 C4 + C6 C1 C4 C6 C4 + C6 WT +++ +++ +++ +++ +++ +++ + − Y27A +++ +++ +++ +++ +++ +++ + − H64A + + + + + + − − H157A − − − − − − − − Y206A + + − − + − − − “+++”: products can be detected; “+”: minor products can be detected; “−”: no products Chen et al. Biotechnol Biofuels (2018) 11:155 Page 12 of 16 β-glucosidase. The two enzymes can alternately hydro - − 3), suggesting that it may function as a carbohydrate- lyze glucuronic acid-containing cello-oligosaccharides to binding module to enhance binding affinity. The evidence yield glucuronic acid [33, 34]. As an important organic that stacking of Tyr203 in LsAA9A (Tyr206 in CtPMO1) acid, glucuronic acid may act as a chelate required for is a major interaction in LsAA9A:Cell5 supports this sug- manganese peroxidase to stimulate its activity by stabiliz- gestion . Mutation of the residue Tyr27 in CtPMO1 3+ ing Mn for depolymerization of lignin , or it may be results in no loss of activity of C1, C4, and C6 oxidation metabolized by the uronic acid pathway in cells to pro- on cellulose, perhaps because the residue Tyr27 is far duce important active substances, such as ascorbate (Vc) away from the copper ion-binding site. Because of the and d-xylulose [32, 36]. absence of structural data of the residue Tyr27, whether Fungal oxidative degradation of cellulose is complex in it plays a role in CtPMO1-substrate interaction needs be nature. Recent genomic sequencing shows multigenic- further studied in the future. ity of AA9 genes in fungi . One of the most extreme It has been suggested that there are different modes for examples is that Coprinopsis cinerea contains 33 putative substrate binding by PMOs, inter-chain binding modes AA9 genes . Two thermophilic fungi iel Th avia ter - and intra-chain binding modes, which allow the active restris and Myceliophthora thermophila contain 24 and site of PMOs to be close to hydrogen of C1, C4, and C6 20 putative AA9 genes, respectively . The thermo - carbon of cellulose . Recent studies of PMO–sub- philic fungus C. thermophilum contains 19 putative AA9 strate interaction show that the active site of LsAA9A genes (http://www.funga lgeno mics.cn). These data sug - from Lentinus similis is close to hydrogen of C1, C4, gest that AA9 proteins have diverse functions, including and C6 carbon of the soluble Cell5 substrate . These regioselectivity diversity. structural data support C1, C4, and C6 oxidation of PMOs on cellulose and cello-oligosaccharides. It is unclear how CtPMO1 oxidizes C6 carbon of cello- Conclusions oligosaccharides. To our knowledge, only galactose CtPMO1 was successfully expressed and correctly pro- oxidase (EC 18.104.22.168) is a single C6-oxidizing copper met- cessed in P. pastoris. A simple and effective chemical alloenzyme that catalyses C6 oxidation of galactose and method to directly identify C4- and C6-oxidized prod- its derivatives [4, 26, 29]. Although the overall sequence ucts by Br oxidation. CtPMO1 can cleave PASC and cel- similarity is low between galactose oxidase and PMO, the loheptaose, and product identification shows that it also two enzymes have very similar active sites. Four highly can oxidize three carbon positions in PASC and cello- conserved amino acid side chains (from Tyr272, Tyr495, oligosaccharides so belonging to a C1-, C4- and C6-oxi- His496, and His581) directly coordinate copper in a dizing PMO. The mutants of CtPMO1 demonstrated that galactose oxidase and the residue Tyr495 is buried and Y27A retained complete activity of C1, C4, C6 oxida- lies in the protein-facing axial position with a distance of tion, indicating Tyr27 effects little to activity of C1, C4, 2.6 Å from the copper to the oxygen atom of the Tyr459 C6 oxidation; Y206A retained partial activity of C1 and side chain , similar to the highly conserved and bur- C4 oxidation but completely lost activity of C6 oxidation, ied residue Tyr168 within CtPMO1. indicating that Tyr206 mainly affects activity of C6 oxida - C6 oxidation of PMOs is interesting because, unlike tion with partial impact on activity of C1 and C4 oxida- C1 and C4 oxidation, it cannot directly cleave the glyco- tion; H64A almost completely lost activity of C1, C4, C6 sidic bond of cellulose. CtPMO1 C6-oxidizes soluble oli- oxidation, indicating His64’s importance in C1, C4, C6 gosaccharides to produce C6-oxidized oligosaccharides oxidation; H157A completely lost activity of C1, C4, C6 (C6-hexodialdose). It is possible that an unknown mech- oxidation, indicating that His157 has a crucial role in the anism (e.g., a secreted oxidoreductase) might further overall activity of CtPMO1. oxidize C6-hexodialdose generated by PMOs to form glu- curonic acid-containing cello-oligosaccharides as I oxi- dizes C6-hexodialdose. We hypothesize that there may Methods be two possible enzymatic reactions to degrade glucu- Strains, plasmids, culture media, and chemicals ronic acid-containing cello-oligosaccharides. One is beta- Chaetomium thermophilum CGMCC3.17990 strain was elimination by polysaccharide lyase. It is well known that previously isolated in China and deposited in the publicly polysaccharide lyase family 20 endo-beta-1,4-glucuronan accessible culture collection CGMCC (Beijing, China). lyases can cleave the glycosidic bond of a glucuronic acid- We purchased the plasmid vector pPICZαA and Pichia containing cello-oligosaccharide via beta-elimination pastoris GS115 strain from Invitrogen. For total RNA [30–32]. The other is hydrolysed by β-glucuronidase and isolation, we grew C. thermophilum at 50 °C for 48 h in Chen et al. Biotechnol Biofuels (2018) 11:155 Page 13 of 16 a medium containing 2% avicel, 0.4% yeast extract, 0.1% protein through affinity chromatography on a His Trap K HPO ·3H O, and 0.05% MgSO ·7H O, dissolved in column (GE Healthcare) with the following steps: bal- 2 4 2 4 2 tap water. Avicel PH-101, glucose, gluconic acid, and anced His Trap column with buffer A (300 mM NaCl, ascorbate were from Sigma-Aldrich. Cellodextrin oligo- 2.7 mM KCl, 10 mM K HPO , 2 mM K H PO , 10 mM 2 4 2 4 saccharide mixture and cellopentaose were from Elicityl imidazole, pH 7.4), then loaded the crude enzyme fol- (Crolles, France). Other reagents were of analytic grade. lowed by rebalancing the column with buffer B (300 mM NaCl, 2.7 mM KCl, 10 mM K HPO , 2 mM K H PO , 2 4 2 4 Molecular cloning of cDNA 30 mM imidazole, pH 7.4), and eluted CtPMO1 protein We used Trizol reagent (Gibco) for total RNA isolation by buffer C (300 mM NaCl, 2.7 mM KCl, 10 mM K HPO , 2 4 of C. thermophilum from mycelia. We performed RT-2 mM KH PO , 250 mM imidazole, pH 7.4). The purified 2 4 PCR with RNA PCR Kit 3.0 instruction (Takara). We protein was pooled and dialyzed fractions overnight at used PCR to amplify the cDNA of the CtPMO1 protein, 4 °C against three changes of 10 mM HAc-NH Ac buffer 2+ termed Ctpmo1, with a pair of specific oligonucleotide (pH 5.0). We used the purified and desalted Cu -loaded primers (CtPMO1-cF/CtPMO1-cR) synthesized based on CtPMO1 protein for further functional studies. the gene (KC441882) from the genomic sequencing of C. thermophilum (Additional file 1: Table S1). Protein determination and SDS‑PAGE We used the Lowry method for protein determination Construction of Ctpmo1 expression vector , determining the purity of the CtPMO1 protein We used PCR to amplify the Ctpmo1 fragment of the using SDS-PAGE . coding region without a signal peptide sequence with a pair of specific primer (CtPMO1-F/CtPMO1-R), which The N‑terminal amino acid sequence analysis of CtPMO1 contained an XhoI and an XbaI restriction site, respec- protein tively (Additional file 1: Table S1). The PCR product was We applied LC–MS/MS to determine the N-terminal digested with XhoI and XbaI and ligated with pPICZαA, amino acid sequence of CtPMO1. We performed in- yielding the expression plasmid pPICZαA/Ctpmo1, gel tryptic digestion of the purified CtPMO1 with the which ensured the expression of CtPMO1 in P. pastoris method previously described , extracting and analyz- with a native N-terminus (Invitrogen). Through DNA ing the resulting peptides with a nano-LC combined with sequencing, we confirmed that the constructed recom - Q Exactive mass spectrometer (Thermo Scientific) in the binant plasmid pPICZαA/Ctpmo1 contained the Ctpmo1 positive ion mode . We acquired MS and MS/MS sequence. spectra on the mass range of m/z range of 300–1800 and 100–1000, respectively. We analyzed all data using MAS- Transformation of P. pastoris COT 2.2 software (Matrix Science) and searched MS/MS After linearized with SacI, we transformed the recombi- spectra against the CtPMO1 protein sequence database. nant plasmid pPICZαA/Ctpmo1 to P. pastoris GS115 by electroporation with BTX ECM830 Electroporator (Har- Activity assay vard Apparatus). We selected the transformants on YPDS We used PASC as substrate, prepared from Avicel plates containing 100 mg/L zeocin and verified them with according to the method previously described . Assays PCR amplifications and DNA sequencing (Invitrogen). contained 5 mg/mL (0.5%) PASC or 1 mg/mL (0.1%) cel- 2+ lopentaose and 5 μM Cu -loaded CtPMO1 protein in CtPMO1 induction and purification 10 mM HAc-NH Ac (pH 5.0) and 1 mM ascorbate for We induced the CtPMO1 protein in transformed P. 48 h at 50 °C. When PASC used as substrate, the reaction pastoris with the Pichia Expression System Kit (Invitro- mixture was centrifuged at 10,000g at 4 °C for 10 min. gen). The transformed P. pastoris was cultured at 28 °C The supernatant was recovered for analysis of soluble for 6 days in a shake flask in BMMY medium containing reaction products of CtPMO1. The precipitate (residual 2+ 1 mM Cu . We centrifuged 1000 mL of the culture fil - PASC) was washed with water three times and was finally trate at 10,000g at 4 °C for 15 min. To the supernatant, suspended in 10 mM HAc-NH Ac (pH 5.0). To release we added (NH ) SO to 90% saturation and gently stirred oxidized oligosaccharides from insoluble reaction prod- 4 2 4 and kept the solution for 12 h at 4 °C. We collected the ucts of CtPMO1, the residual PASC was hydrolyzed with resulting precipitate by centrifuging it at 10,000g at 4 °C endo-1,4-beta-glucanase from Acidothermus cellulolyti- for 15 min, then dissolved it in 50 mM phosphate buff - cus (Sigma) at 50 °C for 10 min, centrifuged at 10,000g at ered saline buffer (pH 7.4), and dialyzed it overnight at 4 °C for 10 min, and the supernatant was recovered for 4 °C against at least three changes of the same buffer. analysis of insoluble reaction products of CtPMO1. We purified the C-terminal histidine-tagged CtPMO1 Chen et al. Biotechnol Biofuels (2018) 11:155 Page 14 of 16 TLC three steps for each cycle (95 °C for 20 s, 55 °C for 20 s, We applied TLC to analyze CtPMO1 products, apply- 72 °C for 2 min) followed by a final elongation step at ing samples to TLC on a Silica gel 60 F254 (Merck). We 72 °C for 5 min. The mutated proteins were expressed developed the plates using ethyl acetate:methanol:acetic and purified as the wild-type CtPMO1 protein. acid:water (4:2:0.25:1, v/v) and visualized CtPMO1 prod- ucts by heating them at 85 °C for 30 min with the chro- Homology modeling mogenic agent, which contained 4 mL phenylamine, 4 g Homology modeling of CtPMO1 was carried out using diphenylamine, 30 mL 85% (w/w) phosphoric acid, and Swiss-Model server (http://www.swiss model .expas y.org). 200 mL acetone. Cellodextrin oligosaccharide mixture Additional file was used as markers. 2+ Additional file 1: Figure S1. SDS-PAGE of the purified Cu -CtPMO1 produced in Pichia pastoris. Figure S2. The N-terminal amino acid MALDI‑TOF MS and MALDI‑TOF MS/MS sequence analysis of CtPMO1 using LC-MS/MS. Figure S3. MALDI-TOF- We analyzed CtPMO1 using MALDI-TOF MS/MS. MS/MS analysis of m/z 525 from MALDI-TOF-MS analysis. Figure S4. Types We applied samples to MALDI-TOF MS/MS on a 5800 of fragmentation of CtPMO1 C4- and C6-oxidized products (m/z 525). Figure S5. H NMR spetra of CtPMO1 soluble reaction products with PASC MALDI-TOF/TOF analyzer (AB SCIEX) and analyzed as substrate in DMSO-d . Figure S6. Sequence alignment of CtPMO1 and them as described previously . For MALDI-TOF MS NCLPMO9C using ClastalW2. Figure S7. Homology model of the catalytic measurements, we used an ionic preparation of 5-chloro- domain of CtPMO1 using SWISS-MODEL. Figure S8. Homology model of CtPMO1 binding with cellopentaose. Figure S9. Identification of the 2-mecapto-benzothiazole (CMBT) and 2, 5-dihydroxy- mutated CtPMO1 soluble reaction products oxidized by Br using with benzoic acid (DHB) as the matrix. 2 µL of the mixture PASC as substrate MALDI-TOF-MS. Table S1. List of primers used for PCR of the samples and the matrix in a 1:1 ratio (v/v) was of the CtPMO1 protein. Table S2. Fragmentation analysis of the peak of DP -2 (m/z 525) according to Additional file 1: Figure S3, S4. deposited on a target plate. The mass spectrometer was operated in the positive ion mode. MS data acquisition mass range was from m/z 500 to 1100. MS/MS data were Abbreviations acquired on the mass range of m/z range of 10–550. PMOs: polysaccharide monooxygenases; CtPMO1: a PMO from Chaetomium thermophilum; PASC: phosphoric acid-swollen cellulose; TFA: trifluoroacetic Fragmentation ion types were nominated as previously acid; AA: auxiliary activity; PCR: polymerase chain reaction; SDS-PAGE: sodium described . dodecyl sulfate polyacrylamide gel electrophoresis; LC–MS/MS: liquid chromatograph-tandem mass spectrometry; TLC: thin-layer chromatography; HPAEC-PAD: high-performance anion exchange chromatography with pulsed Analysis of CtPMO1 products oxidized by Br amperometric detection; DP: degree of polymerization; MALDI-TOF–MS/ MS: matrix-assisted laser desorption/ionization-time-of-flight tandem mass We used saturated bromine water (approximately 3%, spectrometry; LC–MS: liquid chromatograph–mass spectrometry; NMR: w/v) to oxidize CtPMO1 products at 60 °C for 30 or nuclear magnetic resonance; G1: glucose; G2: cellobiose; G3: cellotriose; G4: 60 min and dried under a stream of nitrogen at 40 °C. cellotetraose; G5: cellopentaose; G6: cellohexose; G7: celloheptaose; TMS: tetramethyl silane. Dried samples were then dissolved in water for MALDI- TOF MS analysis. Authors’ contributions DCL and CC designed the study and wrote the paper. CC, JYC, ZGG, MXW and NL performed and analyzed experiments. CC contributed to the preparation NMR spectroscopy of the figures and tables. All authors reviewed the results. All authors read and approved the final manuscript. CtPMO1 products were dissolved in DMSO-d solution and analyzed by NMR spectroscopy. NMR spectra were recorded at 25 °C on an Avance III 400 MHz instrument Acknowledgements Not applicable. (Bruker), using TMS (δ = 0.00) as internal reference. One-dimensional spectra were acquired and processed Competing interests using standard MestReNova software (Bruker). The authors declare that they have no competing interests. Consent for publication Not applicable. Site‑directed mutagenesis Site-directed mutagenesis of CtPMO1 was carried Ethics approval and consent to participate Not applicable. out according to the QuickChangeTM Site-Directed mutagenesis Kit (Stratagene, USA). The sequences of the Funding primers used for CtPMO1 site-directed mutagenesis are This work was supported by the Chinese National Key Technology Support Program (2015BAD15B05), the Chinese National Nature Science Foundation shown in Additional file 1: Table S1. The PCR for muta - (31571949) and the Chinese National Programs for High Technology, Research, tion was performed with the following amplification pro - and Development (2012AA10180402). gram: 1 cycle at 95 °C for 2 min, 20 cycles composed of Chen et al. Biotechnol Biofuels (2018) 11:155 Page 15 of 16 18. Patel I, Kracher D, Ma S, Garajova S, Haon M, Faulds CB, Berrin JG, Ludwig Publisher’s Note R, Record E. 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Published: Jun 5, 2018
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