Expression, Purification, and Characterization of AknX Anthrone Oxygenase, Which Is Involved in Aklavinone Biosynthesis in Streptomyces galilaeus

Abstract

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His 6 tag was efficiently purified to homogeneity by Ni 2؉ affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.Aclacinomycins, which are anthracycline antibiotics produced by Streptomyces galilaeus, show potent antitumor activity by inhibiting complex formation of DNA and topoisomerase II and thus have lower cardiotoxicity than other anthracyclines that inhibit topoisomerase II by stabilization of cleavable complex (8). Aclacinomycin A, also called aclarubicin, has been clinically used in France, Japan, and other Asian countries for the treatment of carcinoma of the stomach, pulmonary carcinoma, oophoroma, malignant lymphadenoma, and acute leukemia. The aglycone moiety of aclacinomycins is aklavinone, which also serves as a common precursor for aglycones of other anthracyclines such as daunomycinone, pyrromycinone, and ε-rhodomycinone, etc. Figure 1 shows the chemical structures of representative anthracycline antibiotics.Previously, we cloned the aklavinone biosynthetic gene cluster from S. galilaeus strain 3AR-33, a mutant strain accumulating aklavinone (18). The 3.4-kb BamHI fragment complemented aklavinone production in the mutant strain ANR-58 as well as aclacinomycin production in the strain . Nucleotide sequence analysis of the fragment showed the presence of open reading frames (ORFs) most typical of bacterial type II polyketide synthase (PKS) genes that code for ␤-ketoacyl synthase (KS), the so-called chain-length factor, and acyl carrier protein (3). In addition to these minimal PKS genes, we noted the presence of a small ORF, aknX, just upstream of the KS gene aknB. Such genes are not found in typical type II PKS gene clusters like that of actinorhodin (2). However, s...

Oxytetracycline Biosynthesis

“…Sequence homology places OxyR within the pyridoxine 5Ј-phosphate oxidase-like protein family, which also includes ActVA-ORF2, an unassigned protein from the actinorhodin gene cluster (69). On the other hand, OxyG is predicted to be a small quinone-forming monooxygenase with homology to enzymes found in several other polyketide clusters such as mithramycin (70), aklavinone (71), and polyketomycin (62). AknX was shown in vitro to catalyze the quinone-forming reaction converting emodin anthrone to emodin, leading the authors to believe that it catalyzes the parallel reaction in the conversion of aklanonic acid anthrone to aklanonic acid (71).…”

“…On the other hand, OxyG is predicted to be a small quinone-forming monooxygenase with homology to enzymes found in several other polyketide clusters such as mithramycin (70), aklavinone (71), and polyketomycin (62). AknX was shown in vitro to catalyze the quinone-forming reaction converting emodin anthrone to emodin, leading the authors to believe that it catalyzes the parallel reaction in the conversion of aklanonic acid anthrone to aklanonic acid (71). In the case of mithramycin, however, inactivation of the OxyG homolog MtmOIII was shown to have no effect on mithramycin production (72).…”

Premithramycinone G, an Early Shunt Product of the Mithramycin Biosynthetic Pathway Accumulated upon Inactivation of Oxygenase MtmOII

“…As shown in Scheme 3, it is necessary that one of these two enzymes, presumably MtmOI, oxidizes the carbon atom in the α-position to the acyl-ACP ester carbonyl group, which eventually becomes the O atom in the 1′-position of 1 (=4-position of 2 and 3, =1-position of 4). MtmOIII normally does not participate in MTM biosynthesis, but might however be responsible for the anthrone oxidations observed in the shunt pathways to 4 and 5 since MtmOIII shows high similarities to anthrone oxygenases, such as AknX (40% amino acid identity, 55% similarity to MtmOIII), involved in aklavinone biosynthesis [40] or HedQ (36% amino acid identity, 51% similarity), the anthrone oxygenase of the hedamycin biosynthesis. [41] In summary, the inactivation of the mtmOII gene resulted in an unexpected metabolite, premithramycinone G (4), which we assume to be a shunt product of the biosynthetic pathway.…”

HypC, the Anthrone Oxidase Involved in Aflatoxin Biosynthesis

“…Oxidation is required for formation of the first stable intermediate, for example, the anthraquinone norsolorinic acid in aflatoxin biosynthesis. Oxidation of bacterial anthrones is catalyzed by a 14-kDa monooxygenase that does not require metal ions, prosthetic groups, or cofactors normally associated with oxygen activation (6,11,16). Previously, an anthrone oxidase that catalyzed the conversion of emodin anthrone to emodin was isolated from cultures of Aspergillus terreus (5, 11), but the gene encoding this protein has not yet been characterized, even though the entire genome of A. terreus has been sequenced (12).…”

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On the basis of gene disruption and enzyme activity, hypC, an open reading frame in the region between the pksA (aflC) and nor-1 (aflD) genes in the aflatoxin biosynthesis gene cluster, encodes a 17-kDa oxidase that converts norsolorinic acid anthrone to norsolorinic acid.Anthraquinones are natural products of polyketide origin that are found in many organisms, including bacteria, fungi, plants, and insects (1,6,11,16). They are the precursors of the highly toxic and carcinogenic aflatoxins (AFs), compounds found as ubiquitous contaminants of maize, cotton seed, and groundnuts (8).

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