ValC, a new type of C7-Cyclitol kinase involved in the biosynthesis of the antifungal agent validamycin A

Abstract

The gene valC, which encodes an enzyme homologous to the 2-epi-5-epi-valiolone kinase (AcbM) of the acarbose biosynthetic pathway, was identified in the validamycin A biosynthetic gene cluster. Inactivation of valC resulted in mutants that lack the ability to produce validamycin A. Complementation experiments with a replicating plasmid harboring full-length valC restored the production of validamycin A, thus suggesting a critical function of valC in validamycin biosynthesis. In vitro characterization of ValC revealed a new type of C7-cyclitol kinase, which phosphorylates valienone and validone--but not 2-epi-5-epi-valiolone, 5-epi-valiolone, or glucose--to afford their 7-phosphate derivatives. The results provide new insights into the activity of this enzyme and also confirm the existence of two different pathways leading to the same end-product: the valienamine moiety common to acarbose and validamycin A.

Figure 1

Inactivation of valC in S. hygroscopicus 5008. A) Schematic representation of the replacement of a 913 bp internal fragment of valC with the 1.4 kb aac(3)IV. In shuttle plasmid pJTU474, aac(3)IV was inserted between 1.54 kb and 1.36 kb genomic fragments originally flanking the 913 bp region. While wild-type 5008 should give a 1.7 kb PCR-amplified product, mutant ZYR-1 should yield a 2.3 kb product using a pair of primers (ValC-F and ValC-R); B) PCR analysis of wild-type 5008 and mutant ZYR-1. PCR products were run on an agarose gel; C) HPLC comparison between cultures of the wild-type strain 5008 and the mutant ZYR-1. The peak corresponding to validamycin A is absent in mutant ZYR-1 cultures; D) Bioassay of fermentation broths of wild-type strain (5008), valC mutant (ZYR-1) and valC complemented mutant (ZYR-1/pJTU704). The agar plugs in the center of each plate are the indicator strain, Pellicularia sasakii. The fermentation supernatant of each sample was mixed with melted agar. The valC mutant did not produce validamycin A but regained the wild-type capability of producing the antibiotic after complementation with the shuttle plasmid pJTU704 carrying the full-length valC.

Figure 2

Heterologous production and characterization of the C₇-cyclitol kinase ValC. A) SDS-PAGE analysis of His₆-tagged ValC protein. Lanes: 1, molecular weight marker; 2, soluble protein of extract of E. coli BL21 Gold(DE3)pLysS/pTMVC-1 before induction; 3, total protein of extract of E. coli BL21 Gold(DE3)pLysS/pTMVC-1 after induction with IPTG; 4, soluble protein of lane 3; 5, purified His₆-tagged ValC; B) Thin-layer chromatography analyses of ValC assay mixtures (solvent system, n-butanol:ethanol:water, 9:7:4). Lanes: 1, ValC without substrate; 2, ValC with 2-epi-5-epi-valiolone and ATP; 3, ValC with 5-epi-valiolone and ATP; 4, ValC with valienone and ATP; 5, ValC with validone and ATP; 6, Boiled ValC with valienone and ATP; 7, ValC with valienone and ATP; 8, ValC with valienone without ATP; 9, ValC with d-glucose and ATP; 10, ValC with d-glucose without ATP; C) and D) Partial ¹H NMR spectra of valienone and valienone 7-phosphate measured on a Bruker DPX-300.

Figure 3

¹H-³¹P COSY of valienone 7-phosphate measured on a Bruker DRX-400. A strong correlation between the H-7 and the phosphorous signals supports the assignment of the phosphorylation site as the C-7 hydroxyl group.

Figure 4

Multiple amino acid sequence alignment of C₇-cyclitol kinases. ValC, valienone/validone kinase (S. hygroscopicus 5008); VldC, glucokinase (?) (S. hygroscopicus var. limoneus); AcbM, 2-epi-5-epi-valiolone kinase (Actinoplanes sp.); AcbK, acarbose kinase (Actinoplanes sp.). ValC, VldC and AcbM have the altered conserved CXCGX(2)GCXE motif (Motif I) and the ATP-binding motif D[ILV]G[GA][T] (Motif II).^[22] However, the last three amino acid residues (CXE) of Motif I are replaced by HLG and HVA in ValC/VldC and AcbM, respectively. AcbK lacks both of these conserved motifs.

Scheme 1

A) Chemical structures of acarbose and validamycin A, B) proposed biosynthetic pathway to validamycin A and functions of ValC/VldC reported by Suh.^[13] VldH was proposed to be a phosphomutase.

Scheme 2

Synthesis of valienone and its conversion to valienone 7-phosphate catalyzed by ValC.

Scheme 3

The PK/LDH coupled enzyme assay system used in the kinetic analysis of ValC.

Scheme 4

Proposed biosynthetic pathways to the valienamine moiety of acarbose and validamycin A