Send Orders for Reprints to [email protected] 10 Protein & Peptide Letters, 2014, 21, 10-14 Medium-Chain Dehydrogenases with New Specificity: Amino Mannitol Dehydrogenases on the Azasugar Biosynthetic Pathway Yanbin Wu, Jeffrey Arciola, and Nicole Horenstein* Department of Chemistry, University of Florida, Gainesville Florida, 32611-7200, USA Abstract: Azasugar biosynthesis involves a key dehydrogenase that oxidizes 2-amino-2-deoxy-D-mannitol to the 6-oxo compound. The genes encoding homologous NAD-dependent dehydrogenases from Bacillus amyloliquefaciens FZB42, B. atrophaeus 1942, and Paenibacillus polymyxa SC2 were codon-optimized and expressed in BL21(DE3) Escherichia coli. Relative to the two Bacillus enzymes, the enzyme from P. polymyxa proved to have superior catalytic properties with a Vmax of 0.095 ± 0.002 mol/min/mg, 59-fold higher than the B. amyloliquefaciens enzyme. The preferred substrate is 2- amino-2-deoxy-D-mannitol, though mannitol is accepted as a poor substrate at 3% of the relative rate. Simple amino alco- hols were also accepted as substrates at lower rates. Sequence alignment suggested D283 was involved in the enzyme’s specificity for aminopolyols. Point mutant D283N lost its amino specificity, accepting mannitol at 45% the rate observed for 2-amino-2-deoxy-D-mannitol. These results provide the first characterization of this class of zinc-dependent medium chain dehydrogenases that utilize aminopolyol substrates. Keywords: Aminopolyol, azasugar, biosynthesis, dehydrogenase, mannojirimycin, nojirimycin. INTRODUCTION are sufficient to convert fructose-6-phosphate into manno- jirimycin [9]. We proposed that the gutB1 gene product was Azasugars such as the nojirimycins [1] are natural prod- responsible for the turnover of 2-amino-2-deoxy-D-mannitol ucts that are analogs of monosaccharides that feature a nitro- (2AM) into mannojirimycin as shown in Fig. 2. Feeding the gen in the ring rather than oxygen (Fig. 1). In Nature they are B. amyloliquefaciens gabT1 knockout with 2AM restored produced by various Bacillus, Streptomyces and plant spe- azasugar production [9]. cies [2-4]. Azasugars have served as the inspiration for the synthesis of many different glycosidase inhibitors [5] and Sequence analyses indicate that GutB1 is a zinc- have in recent times also enjoyed applications as chemical dependent NAD(P)-dependent alcohol dehydrogenase, simi- chaperones for assisting in the folding and stabilization of lar to sorbitol dehydrogenase, and is a member of the me- mutant enzymes responsible for lysosomal storage diseases dium-chain dehydrogenase/reductase (MDR) superfamily [6]. Examples include Miglustat, N-butyl-1-deoxynojiri- [11]. As it was known that other species with sequenced ge- mycin, for type I Gaucher’s disease [7] and Miglitol, (N-2- nomes were deoxynojirimycin producers, we sought to iden- hydroxyethyl)-1-deoxynojirimycin for modulation of post- tify and then compare other homologues possessing the prandial bloodsugar in treatment of type II diabetes [8]. unique dehydrogenase activity demonstrated by the B. amy- loliquefaciens enzyme. A protein BLAST analysis of the Although azasugars have been known for quite some three-gene azasugar biosynthetic signature found in B. amy- time, the enzymatic machinery responsible for their synthesis loliquefaciens was conducted and identified similar ORFs in has only recently become the topic of experimental inquiry. Bacillus and related species. When we included more distant Further, the identity of enzymes in the entire pathway has not hits we identified that Paenibacillus polymyxa SC2 has five yet been determined nor have any been characterized indi- ORFs that appear to include the three functions we identified vidually. In this Letter we wish to report the expression and in B. amyloliquefaciens, as well as an additional two functional characterization of a key dehydrogenase found in (PPSC2_c2587 and PPSC2_c2588) that code for putative azasugar biosynthetic clusters. Recent investigation of 1- mannitol dehydrogenase and mannonate dehydratase activi- deoxynojirimycin biosynthesis has led to the identification of ties that may also be part of the pathway [12]. In the work Bacillus amyloliquefaciens genes gabT1, yktC1 and gutB1 as we describe here, we characterize the GutB1 dehydrogenase part of the overall azasugar biosynthetic pathway [9,10]. from B. amyloliquefaciens FZB42, the closely related en- These three genes respectively encode for transaminase, zyme from B. atrophaeus 1942 [13] (86% identity) and the phosphatase and dehydrogenase activity that we have shown more distantly related homolog from Paenibacillus polymyxa SC2 (33% identity). We show that the so-called GutB1 en- *Address correspondence to this author at the Department of Chemistry, zymes prefer substrates bearing an amino group, distinguish- University of Florida, Gainesville Florida, 32611-7200, USA; Tel: 01-(352)- ing themselves from previously characterized polyol dehy- 392-9859; E-mail: [email protected] drogenases. Wu, Y. Horenstein, N. Unpublished observations for recombinant YktC1 from B. amyloliquefaciens. 1875-5305/14 $58.00+.00 © 2014 Bentham Science Publishers Medium-Chain Dehydrogenases with New Specificity Protein & Peptide Letters, 2014, Vol. 21, No. 1 11 Figure 1. Structures of nojirimycin (NJ), mannojirimycin (MJ) and deoxynojirimycin (DNJ). Both NJ and DNJ have the gluco configuration and MJ has the manno configuration. HO HO HO HO HO HO NH OH GutB1 HO HO O HO OH + HO NH HO NH2 NAD NADH 2 HO MJ Figure 2. Conversion of 2-amino-2-deoxy-D-mannitol to mannojirimycin via GutB1 catalyzed oxidation. After oxidation, the acyclic ami- noaldehyde is able to spontaneously cyclize to mannojirimycin (MJ). linker sequence as described above for the B. amyloliquefa- MATERIALS AND METHODS ciens gutB1 construct. General. The gutB1 genes were synthesized and codon P. polymyxa D283N mutation. This point mutation was optimized by GenScript. Escherichia coli DH5alpha and E. made using the Q5 mutagenesis kit from New England Bio- coli BL21 (DE3) strains were obtained from Invitrogen and labs. The forward mutagenic primer sequence was 5’- Novagen respectively. The pET30a expression vector was GAAGATTATCGGCTCAATTAACTCGCTGGGTACC- obtained from Novagen. Restriction endonucleases, T4 DNA TTCTC-3’. The reverse primer sequence was 5’-AGGCTG ligase and thermostable polymerases were purchased from CGATCAACCACTTCTTTCGGATTA-3’. The PCR ther- New England Biolabs. Isopropyl- -D-thiogalactopyranoside mocycle was: 98 °C, 30 s; 25x (98 °C, 10 s; 66 °C, 30 s; 72 (IPTG), kanamycin and protein MW standards were pur- °C, 198 s), 72 °C, 120 s; followed by 4 °C, indefinite. chased from Fisher Scientific. Oligonucleotides were pur- chased from Integrated DNA Technologies. DNA sequenc- Protein expression and purification. The pET30a- ing was performed at the University of Florida Sanger Se- gutB1constructs were transformed into BL21 (DE3) E. coli. quencing core facility. Single colonies were selected to inoculate 5 mL overnight starter cultures (Luria-Bertani medium containing 50 g/mL Subcloning of the B. amyloliquefaciens gutB1 gene kanamycin). Each starter culture was used to inoculate 500 into pET30a. The codon-optimized version of the gutB1 ml of LB/K medium and the resulting cultures were grown gene was originally prepared in pETBlue2. The pETBlue2- for 2-4 hours at 37 °C with constant shaking at 225 rpm. gutB1 vector served as a template for PCR with the forward Expression of GutB1 was induced by the addition of 0.5 mM primer 5’-GAGCCATGGGGATGAAAGCTCTGGTGTG IPTG when an OD600 of 0.4 to 0.8 was reached. The cultures GAC-3’ and reverse primer 5’-AGAGCTCGAGTTACAGC were grown at 18 °C for 18 h with constant shaking at 225 AGTTTCGGGTCGCTAAC-3’. The underlined sequences rpm. The cells were harvested by centrifugation at 4 °C in a correspond to the NcoI and XhoI restriction sites, in the for- GS3 rotor at 5000 rpm for 20 minutes. The cell pellets were ward and reverse primers, respectively. The PCR product each resuspended in 20 mL of lysis buffer (20 mM Tris-HCl, was cloned into pET30a to generate the reconstruct plasmid pH=7.9, 250 mM NaCl, 5 mM imidazole). After lysis by pET30a-opt-gutB1, and was verified by sequencing. This French press and sonication, the lysate was centrifuged at construct codes for an N-terminal Met(His)6 tag fused to a 14,000 rpm for 30 min at 4 °C in an SS34 rotor. The clarified linker peptide with the following sequence upstream of the lysate was applied to a Ni-IMAC resin (Qiagen), and the native start Met: SSGLVPRGSGMKETAAAKFERQHMDS column washed with a step gradient of 10, 50, and 100 mM PDLGTDDDDK. imidazole. GutB1 was eluted with 250 mM imidazole- Expression constructs for P. polymyxa SC2 and B. containing buffer (20 mM Tris-HCl, pH=7.9, 250 mM NaCl, atrophaeus gutB1 genes. The P. polymyxa (locus tag PPSC2 250 mM imidazole). The eluted enzyme was dialyzed at 4°C _c2584) and B. atrophaeus (locus tag BATR1942_19425) against 20 mM Tris buffer (50 mM NaCl, pH=7.0). gutB1 homologues were codon-optimized (Genscript) in Protein analyses. Protein was assayed by the Bradford pUC57 with NcoI and XhoI sites flanking the start and stop method using bovine serum albumin as a standard [14]. codons. The gutB1 genes were subcloned into the NcoI and SDS-PAGE was performed at room temperature using 12% XhoI sites of the pET30 vector for expression. The recombi- acrylamide resolving gel and 5% acrylamide stacking gel. nant construct was confirmed by sequencing. These con- Metal content was determined by inductively coupled structs afforded coded for the same N-terminal His tag and 12 Protein & Peptide Letters, 2014, Vol. 21, No. 1 Wu et al. plasma-mass spectrometry (ICP-MS) at the Chemical Analy- RESULTS AND DISCUSSION sis Laboratory, University of Georgia, Athens. Samples were The three GutB1 dehydrogenase genes from B. amyloliq- exchanged into Chelex-treated 20 mM Tris HCl pH 7.0 uefaciens FZB42 (Bam), B. atrophaeus 1942 (Bat), and P. buffer. Approximately 2.5 – 3 mg of protein in metal-free polymyxa SC2 (Ppo) were obtained by synthesis in codon buffer was further incubated with Chelex for 2 h at 4 °C to remove weakly bound metals.