Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061
- Tao Zhang1, 2,
- XiaoPeng Jia1, 2,
- Ying Zhuo1,
- Mei Liu1,
- Hong Gao1,
- JinTao Liu^1, 2 and
- Lixin Zhang1Email author
© Zhang et al.; licensee BioMed Central Ltd. 2012
Received: 13 June 2012
Accepted: 15 August 2012
Published: 23 August 2012
The ketoisovalerate reductase (EC 22.214.171.124 ) is required for the formation of beauvericin via the nonribosomal peptide synthetase biosynthetic pathway. It catalyzes the NADPH-specific reduction of ketoisovaleric acid to hydroxyisovalerate. However, little is known about the bioinformatics’ data about the 2-Kiv reductase in Fusarium. To date, heterologous production of the gene KivRFp from Fusarium has not been achieved.
The KivRFp gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene KivRFp contained a 1,359 bp open reading frame (ORF) encoding a polypeptide of 452 amino acids with a molecular mass of 52 kDa. Sequence analysis indicated that it showed 61% and 52% amino acid identities to ketoisovalerate reductase from Beauveria bassiana ATCC 7159 (ACI30654) and Metarhizium acridum CQMa 102 (EFY89891), respectively; and several conserved regions were identified, including the putative nucleotide-binding signature site, GXGXXG, a catalytic triad (Glu405, Asn184, and Lys285). The KivRFp exhibited the highest activity at 35°C and pH 7.5 respectively, by reduction of ketoisovalerate. It also exhibited the high level of stability over wide temperature and pH spectra and in the presence of metal ions or detergents.
A new ketoisovalerate reductase KivRFp was identified and characterized from the depsipeptide-producing fungus F. proliferatum. KivRFp has been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering and directed evolution, towards the goal of developing efficient enzyme for downstream biotechnological applications.
Keywords2-Kiv Reductase Beauvericin Fusarium proliferatum LF061
In mammals, the catabolism of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine proceeds by two-step process, i.e. transamination step (aminotransfer) and oxidation step. BCAAs are usually transaminated to form 2-keto acids by branched-chain amino acid transaminases (BCATs). These 2-keto acids are oxidatively decarboxylated by mitochondrial branched-chain 2-ketoacid dehydrogenase [13–15] and converted to acyl CoA. However, in some fungal species, namely the hydroxysiovalerate-containing CODs producers, there is another pathway leading from L-Val to D-Hiv via the keto acid (Figure 1). D-hydroxyisovalerate dehydrogenase, catalyzes the reversible reduction of 2-ketoisovalertate (2-Kiv), the intermediate of branched-chain amino acid metabolism , to D-Hiv in the presence of NADPH, which cannot be replaced by NADH [17–19]. So far, D-HivDH has been purified and partially characterized exclusively from the enniatin producer, Fusarium sambucinum[17, 19], but the corresponding protein and gene sequences were not determined. Xu et al., demonstrated the novel kivr-encoding 2-ketoisovalerate reductase is the sole supplier of D-Hiv for both beauvericin and bassianolide biosynthesis in Beauveria bassiana ATCC 7159 .
On the other hand, little is known about the bioinformatics’ data about the 2-Kiv reductase (2-KivR) in Fusarium, thus warranting further study. To date, heterologous production of the gene KivRFp from Fusarium has not been achieved; similarly, very little information is available about the role of the KivRFp in the regulation of beauvericin biosynthesis. In order to get a closer insight into the unique and intriguing features of the ketoisovalerate reductase in the Fusarium genus, we undertook both genetic and biochemical studies. In the present article, we describe the identification and functional analysis of the KivRFp of Fusarium proliferatum LF061. Our report demonstrates that the KivRFp constituted a new member of family ketonate oxidoreductases.
Chemicals were of the highest purity commercially available. Pyruvate, 2-Kiv, 2-ketovalerate, 2-ketoglutarate and 2-ketobutyrate were obtained from TCI Japan Ltd. D-Hiv were purchased from Sigma. NADH, NADPH, NADP+ were products of Roche.
Bacterial strains, plasmids, and culture
Starting bacterial strains and plasmids used in this study
Strain or plasmid
E. coli TOP10
lacх74 recA1 deoR F – mcrA Δ (mrr-hsdRMS-mcrBC) ϕ80 lacZΔM15Δ araD139Δ (ara-leu)7697 galU galK
E. coli BL21(DE3)
F–, ompT, hsdSB (rB–, mB–), dcm, gal, λ(DE3), pLysS, Cmr
Fusarium proliferatum LF061
Wild strain, producing beauvericin isolated from marine sample
E. coli TOP10-KivRFp
Positive clone of artificial gene synthesis, which carries the KivRFp gene fragment
E. coli BL21(DE3)-KivRFp
Positive clone, which carries the pKivRFp-His expression vector
Cloning vector; Apr
Expression vector; Kmr
pUCE, which carries the complete ketoisovalerate reductase (KivRFp)
pET28a carrying amplified EcoRI-NdeI fragment containing ketoisovalerate reductase gene (KivRFp)
DNA preparation and manipulation
E. coli cells were transformed by the calcium chloride procedure . Recombinant plasmid DNA was isolated by the method of Birnboim and Doly . For sequencing, this DNA was further purified by polyethylene glycol precipitation . Restriction enzymes, T4 DNA ligase and calf intestinal alkaline phosphatase were purchased from New England Biolabs (Ipswich, USA) or Takara (Tokyo, Japan) and used according to the manufacturers’ instructions. BugBuster Ni-NTA His. Bind Purification Kit was purchased from Novagen (Code No. NV70751-3, Novagen).
Deduced amino acid sequences of 13 ketonate reductases or homologous proteins were subjected to protein phylogenetic analysis. A phylogenetic tree was generated using the neighbor joining method of Saitou and Nei  with MEGA 4.0 software . A total of 4 sequences were aligned with the CLUSTAL_W program  and visually examined with BoxShade Server program. The length of each branch pair represents the evolutionary distance between the sequences.
Heterologous expression of gene KivRFpand purification of recombinant KivRFp
Artificial gene synthesis was finished by Innovogen Tech. Co. (Beijing, China). Jcat (Java Codon Adaptation Tool)  and OPTIMIZER  were used to improve heterologous protein production. The integrity of the nucleotide sequence of all newly constructed plasmids was confirmed by DNA sequencing. The primer pairs with restriction enzyme sites for HindIII and NdeI were designed to generate an N-terminal His-tag of the recombinant ketonate reductase. The modified KivRFp gene was cloned into an expression vector, pET28a (+) and the recombinant plasmid pKivRFp-His was transformed into E. coli BL21 (DE3) cells. When the cell density at 600 nm reached around 0.6, expression of recombinant KivRFp protein was initiated by addition of 0.6 mM isopropylthio-β-D-galactoside and continued cultivation for additional 6 h at 20°C. Cells were harvested by centrifugation at 6,000 × g for 5 min, washed twice with ice-cold 50 mM sodium phosphate buffer (pH 8.0) and resuspended in the same buffer containing 10 mM imidazole, disrupted by sonification in an ice-water bath (60 times, 5 s). Recombinant KivRFp reductase was applied to metal-chelating chromatography using Ni-NTA affinity chromatography (Novagen) according to the manufacturer’s instructions.
Polyacrylamide gel electrophoresis of enzyme in the presence of sodium dodecyl sulfate (SDS) was carried out by the method of Sambrook and Russell . Protein concentrations were determined by a modified Bradford procedure with bovine serum albumin as a standard .
The standard 2-Kiv reductase assay mixture contained 50 mM sodium phosphate buffer (pH 7.0), 0.7 mM 2-Kiv, 0.29 mM NADPH, and enzyme in a final volume of 3.0 mL. The reaction was initiated by the addition of substrate, and the decrease in absorbance at 340 nm (A340) was measured at 35°C, which was performed using an UNICO 2802 UV/VIS spectrophotometer. A molar extinction coefficient of 6.22 cm2/pmol NADPH was used for the calculation of enzyme activity. One unit was defined as the amount of enzyme caused the oxidation of 1 μmol of NADPH per min. The reaction mixture for the assay of the reverse reaction contained 50 mM sodium phosphate buffer (pH 8.0), 2.8 mM NADP+, 5.7 mM D-Hiv, and enzyme in a final volume of 3.0 mL. The increase in the rate of reduction of NADP+ due to oxidation of D-Hiv was measured at 340 nm (45°C) [18, 19, 29].
Characterization of recombinant KivRFpand biochemical properties
The purified KivRFp was subjected to a series of biochemical analysis, including determine the pH optimum, temperature optimum, and effects of various detergents and metal ions. All measurements were carried out in triplicate. The values were the mean of the data. The substrate specificity of the purified KivRFp protein was performed using the following substrates: pyruvate, 2-Kiv, 2-ketovalerate, 2-ketobutyrate, 2-ketocapronate and 2-ketoglutarate.
The optimum temperature of purified KivRFp was determined by assaying reductase activities in a 50 mM sodium phosphate buffer (pH 8.0) for a temperature range of 25-65°C, in which 2-Kiv (0.7 mM) acted as substrate. Optimal pH was determined by examining the activity of the enzyme after incubation at 40°C for 10 min using 2-Kiv (0.7 mM) as substrate. The buffers used were: 50 mM sodium phosphate buffer (pH 5.0-8.0), 50 mM Tris–HCl buffer (pH 8.0-10.5).
Various metal ions (CoCl2, CaCl2, ZnCl2, MgCl2, K2SO4, FeSO4, CuCl2, MnCl2, and FeCl3), and chelating agent EDTA at final concentrations of 5 mM were added to the enzyme in 50 mM sodium phosphate buffer (pH 8.0), that was assayed for 2-Kiv reductase activity following preincubation at 40°C. Effect of detergents on reductase activity was determined by incubating the enzyme for 10 min at 40°C in 50 mM sodium phosphate buffer (pH 8.0), containing Tween-20, Tween-80, Triton X-100, β-mercaptoethanol, sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), diethylpyrocarbonate (DEPC), phenylmethanesulfonyl fluoride (PMSF), dimethyl sulfoxide (DMSO). The concentrations of metal ions, EDTA, detergents, and surfactants used were 5 mM, 3 mM, and 0.5% (v/v), respectively. The activity of the enzyme preparation in the absence of metal ions and detergents before incubation was defined as the 100% level.
Nucleotide sequence accession number
The DNA sequence of KivRFp from Fusarium proliferatum LF061 was deposited in GenBank under accession number of JQ922252.
Cloning and sequence analysis of KivRFp
We have recently cloned and functionally characterized beauvericin biosynthetic locus (bea) of the F. proliferatum LF061 genome (unpublished data). The FpBEAS gene encoding the beauvericin synthetase was found to be clustered with a gene (KivRFp) encoding a deduced putative 2-ketoisovalerate reductase (KivRFp). Amino acid sequence alignment indicated that KivRFp exhibited low identity with other ketopantoate reductases or hypothetical proteins in GenBank. KivRFp shared the highest sequence identity with the KIVR from B. bassiana ATCC 7159 (ACI30654, 61% identity/75% similarity), followed by the putative 2-ketopantoate reductase from Metarhizium acridum CQMa 102 (EFY89891, 56% identity/70% similarity). Similarity was also detected with the hypothetical or putative 2-dehydropantoate reductases from Gibberella zeae PH-1 (anamorph: F. graminearum) and F. oxysporum Fo5176 (EGU84839, 49% identity; and EGU75687, 47% identity, respectively), and also with functionally uncharacterized terminal reductase domain of the Xylaria sp. bassianolide synthetase (ABR28366, 30% identity/48% similarity).
Expression and purification of recombinant KivRFp
Substrate and cofactor specificity of KivRFp
Effect of temperature and pH on KivRFp
Effect of metal ions on KivRFp
Effect of metal ions on esterase activity
Relative activity (%)
100.0 ± 1.7
16.4 ± 2.9
123.2 ± 3.4
4.9 ± 1.6
7.8 ± 2.7
6.2 ± 1.3
87.7 ± 3.2
7.9 ± 3.4
169.1 ± 3.7
3.7 ± 1.8
195.6 ± 2.7
Effect of detergents and enzyme inhibitors on KivRFp
Effect of detergents and enzyme inhibitors on esterase activity
Relative activity (%)
100.0 ± 2.1
126.9 ± 4.9
42.7 ± 2.2
113.6 ± 2.7
53.6 ± 4.1
6.3 ± 1.9
101.7 ± 2.6
60.8 ± 4.6
176.4 ± 2.1
128.7 ± 3.3
Ketoisovalerate reductases are types of oxidoreductases which are widely distributed from prokaryotes to eukaryotes and which are involved in primary and second metabolism [17, 20, 34–36]. 2-hydroxyisovalerate is a common 2-hydroxycarboxylate constituent of depsipeptides. Reduction of Kiv in fungi provides Hiv, a precursor for the biosynthesis of nonribosomal depsipeptides and aroma compounds. Fungal cyclooligomer depsipeptides synthetase A domains incorporate free 2-hydroxycarboxylate precursors for the synthesis of CODs, preformed by dissociated, independent, monofunctional, NADPH-dependent enzymes with high chiral specificity, exemplified by the D-Hiv dehydrogenase purified from the enniatin producer F. sambucinum (teleomorph: G. pulicaris) [17, 19]. In contrast, known bacterial depsipeptide synthetases employ A domains that activate and load 2-ketocarboxylates as β-ketoacyl thioesters onto their cognate T domains, and utilize integrated ketoacyl reductase domains for the synthesis of 2-hydroxycarboxylate thioesters. This might suggest the biosynthesis of 2-hydroxycarboxylate precursors in bacteria and fungi may represent example of convergent evolution . This enzyme, which plays a key role in beauvericin biosynthesis, catalyzes the reversible reaction of 2-Kiv to D-Hiv using NAD(P)H as a cofactor. 2-ketoisovalerate reductase differs from other NADPH-dependent oxidoreductases with broad substrate specificity by its high affinity for 2-Kiv (Figure 5A). The high specificity of KivRFp may also explain the fact that D-Hiv is the exclusive hydroxy acid component in beauvericin isolated from Fusaria (Logrieco A et al., 1998). To our knowledge this is the first report about the heterologous production of a ketoisovalerate reductase with higher substrate specificity for 2-Kiv.
KivRFp, which was cloned, expressed, and purified, was subjected to molecular and biochemical experiments in this study. The F. proliferatum KivRFp exhibits high amino acid sequence similarity to B. bassiana KIVR and deduced dehydropantoate reductases of fungi, together with low but significant sequence similarity predicted secondary structure to prokaryotic ketopantoate reductases such as PanE of E. coli[20, 30, 31, 36]. KivRFp and the related B. bassiana KIVR are predicted to share the N-terminal αβ-Rossmann fold and the C-terminal α-helical catalytic domain architecture of the E. coli ketopantoate reductase. In the amino acid sequence of KivRFp, the conserved catalytic triad (Glu-Asn-Lys) and substrates stabilization active amino acids of ketonate oxidoreductases are still the same. In ketopantoate reductase PanE from E. coli, the protonated Lys176 (KivRFp: Lys285, Figure 2) could function as a general acid to polarize the carbonyl in the direction of ketopantoate reduction, facilitates the hydride transfer from NADPH to the C2 carbonyl of ketopantoate, and protonates the intermediate alkoxide [20, 31]. Asn98 (KivRFp: Asn184) forms hydrogen binding both with the carboxylate of the ketopantoate and with the ribose of the nicotinamide: this orients the cofactor. And also, Glu256 (KivRFp: Glu405) and Lys72 (KivRFp: Lys158) further participate substrate binding to the NADPH . Further stabilization is provided by a hydrogen bond between the Ser244 (KivRFp: Ser393) and carboxyl group of the ketopantoate, and between Asn180 (KivRFp: Asn289) and the a-hydroxy group of the product. And also, KivRFp showed sequence and the structural similarity with ketopantoate reductases, but not with D-lactose or D-hydroxyisocaproate dehydrogenase . Further biochemical assay demonstrated that Fusarium KivRFp could utilize NADPH and NADH as coenzyme, while the NADPH is preferred (Figure 5B). In accordance to our results, bacterial chiral enzymes belonging to the D-ketoacid reductases superfamily also could utilize NADH as coenzyme, and also feature a conserved Arg-(Asp/Glu)-His active site triad [20, 37]. Based on the phylogenetic, molecular, and biochemical analysis results, we propose that KivRFp is a new member of family ketonate oxidoreductases. Future work will establish the structure of this enzyme to gain more information about its catalytic mechanism.
In conclusion, we identified a new ketoisovalerate reductase KivRFp from the depsipeptide-producing fungus F. proliferatum. KivRFp is expected to show high potential for downstream biotechnological applications including synthetic organic chemistry and combinatorial biosynthesis beauvericin homologues. This was confirmed by its extensive biochemical characterization, which revealed the enzymes substrate specificity, wide pH and temperature spectra, and also, stability towards addictives including metal ions and detergents. Enlargement of the ketoisovalerate reductase pool can be an immediate source of genetic modification, or yield enzymes that can be further specialized by directed evolution, and also, this would optimize their industrial applications.
TZ, XPJ, YZ, ML, HG and LXZ, Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, China. JTL, deceased.
Nonribosomal peptide synthetases
Cyclooligomer depsipeptides synthetase
The authors are very grateful to Elizabeth Ashforth for critical reading and helpful discussions. This work was supported in part by grants from National Natural Science Foundation of China (31100075, 81102362, 31170095, 31000004), the CAS Pillar Program (XDA04074000) and the Ministry of Science and Technology of China (2011ZX11102-011-11, 2007DFB31620,). L. Z. is an Awardee for National Distinguished Young Scholar Program in China.
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