A comparative analysis of the sugar phosphate cyclase superfamily involved in primary and secondary metabolism

Abstract

Sugar phosphate cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, for example, aromatic amino acids, and clinically relevant secondary metabolites, for example, aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically labeled cyclitols revealed that cetoniacytone A, a unique C(7)N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2-epi-5-epi-valiolone, a product of SPC. By using heterologous probes from the 2-epi-5-epi-valiolone synthase class of SPCs, an SPC homologue gene, cetA, was isolated from the cetoniacytone producer. cetA is closely related to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actinoplanes sp. strain A40644. Recombinant expression of cetA and BE-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-5-epi-valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that might regulate the biosynthesis of a novel set of secondary metabolites.

Figure 1

Screening of Cosmid Library for AcbC Homologs. (A) Restriction analysis and corresponding (B) Southern analysis of AcbC positive cosmids. (C) Graphic representation of the three overlapping cosmids, pCET-25, pCET-26, and pCET27.

Figure 2

Phylogenetic analysis of the superfamily of sugar phosphate cyclases. The Phylip software package was used to generate an unrooted maximum likelihood tree. E. coli glycerol dehydrogenase was used as an out-group and support for each node was evaluated with 100 bootstrap replicates of a heuristic search with two random stepwise addition sequences for each replicate. Species information and GenBank accession numbers are indicated for each sample.

Figure 3

Recombinant expression and characterization of the CetA and BE-Orf9 2-epi-5-epi-valiolone synthases. GC-MS profiles of silylated-cyclitol metabolites produced cell-free lysates containing (A) the pRSET-B vector alone, (B) pRSET-B-cetA, or (C) pRSET-B-BE-orf9. (D) GC-MS trace of silylated 2-epi-5-epi-[6,6-²H₂]-valiolone. (A-D) Left panel represent the GC trace and the right panel represents the MS fragmentation pattern of the major peak eluting at 8.65-9.00. (E) SDS-PAGE analysis and Coomassie staining of heterologously expressed and purified CetA and BE-Orf9. Lanes 1 and 3 represent total cell lysate containing CetA or BE Orf9, respectively, Lanes 2 and 4 represent nickel-agarose purified CetA and BE-Orf9, respectively. TLC analysis analyzing the substrate specificity of (F) CetA or (G) BE-Orf9: Enzyme reactions contained 5 mM of the following substrates, Lane 1 = amino-DAHP, Lane 2 = DAHP, Lane 3 = glucose 6-phosphate, Lane 4 = fructose 6-phosphate, Lane 5 = mannose 6-phosphate, Lane 6 = ribose 5-phosphate, Lane 7 = sedo-heptulose 7-phosphate, and Lane 8 = 2-epi-5-epi-valiolone standard. Arrows indicate 2-epi-5-epi-valiolone.

Scheme 1

The diversity of chemical structures derived from sugar phosphate intermediates. The cyclitol core units are shown in bold.

Scheme 2

Biosynthetic Pathways that Parallel the Initial Steps in Shikimate Biosynthesis. DOS= 2-deoxy-scyllo-inosose synthase, EVS= 2-epi-5-epi-valiolone synthase, DHQS= dehydroquinate synthase, aDHQS= aminodehydroquinate synthase.

Scheme 3

Summary of cetoniacytone biosynthetic feeding experiments

Scheme 4

Proposed biosynthetic mechanism for cyclitol formation during BE-40644 biosynthesis.