Crystallization and preliminary X-ray crystallographic study of a methyltransferase involved in 2-methylisoborneol biosynthesis in<i>Streptomyces lasaliensis</i>

Ariyawutthiphan, Orapin; Ose, Toyoyuki; Tsuda, Muneya; Gao, Yong‐Gui; Yao, Ming; Minami, Atsushi; Oikawa, Hideaki; Tanaka, Isao

doi:10.1107/s1744309110051523

Cited by 5 publications

(8 citation statements)

References 12 publications

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“…The procedures used to prepare the proteins and crystals have been described previously (Ariyawutthiphan et al, 2011). We used three types of crystals in this study, i.e.…”

Section: Structure Determinationmentioning

confidence: 99%

“…apo, a complex with SAM and a tertiary complex with both SFG and GPP (hereafter called the substrate complex), to analyze how this enzyme recognizes the substrates and its reaction mechanism. Methods for solving the structures have previously been published (Ariyawutthiphan et al, 2011); the data-collection, processing and structure-refinement statistics are summarized in Table 1. All data sets were integrated, merged and scaled using HKL-2000 (Otwinowski & Minor, 1997).…”

Section: Structure Determinationmentioning

confidence: 99%

“…5b). Attempts to generate cocrystals with only GPP failed and crystals from a mixture of GPPMT and GPP gave only the apo-type structure (Ariyawutthiphan et al, 2011). However, the crystal structure of the substrate-bound form provides many insights into the incorporation of the substrate into the active site.…”

Section: Ordered Substrate Incorporationmentioning

confidence: 99%

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Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependentC-methylation

Ariyawutthiphan¹,

Ose²,

Minami³

et al. 2012

Acta Crystallogr D Biol Cryst

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In the typical isoprenoid-biosynthesis pathway, condensation of the universal C(5)-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C(10)-C(20)). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

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“…The procedures used to prepare the proteins and crystals have been described previously (Ariyawutthiphan et al, 2011). We used three types of crystals in this study, i.e.…”

Section: Structure Determinationmentioning

confidence: 99%

Section: Structure Determinationmentioning

confidence: 99%

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Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependentC-methylation

Ariyawutthiphan¹,

Ose²,

Minami³

et al. 2012

Acta Crystallogr D Biol Cryst

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“…The preliminary X-ray crystallographic analysis of the S. lasaliensis 2-MeGPP synthase has recently been reported. 41 A new profile HMM for the region around two conserved metal-binding motifs of microbial terpene synthases also identified a single open reading frame of 397 aa within the genome sequence of the cyanobacterium, Pseudanabaena limonetica . 42 Heterologous expression of the corresponding gene product has confirmed the identity of the cyanobacterial 2-methylisoborneol synthase as well as the coupled 2-MeGPP synthase.…”

Section: Case Studiesmentioning

confidence: 99%

Exploration and Mining of the Bacterial Terpenome

2011

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CONSPECTUS Tens of thousands of terpenoids are present in both terrestrial and marine plants as well as fungi. In the last 5–10 years, however, it has become evident that terpenes are also produced by numerous bacteria, especially soil-dwelling Gram-positive organisms such as Streptomyces and other Actinomycetes. Although some microbial terpenes, such as geosmin—the degraded sesquiterpene responsible for the smell of moist soil, the characteristic odor of the earth itself—have been known for over 100 years, few terpenoids have been identified by classical structure- or activity-guided screening of bacterial culture extracts. In fact, the majority of cyclic terpenes from bacterial species have only recently been uncovered by the newly developed techniques of “genome mining.” In this new paradigm for biochemical discovery, bacterial genome sequences are first analyzed with powerful bioinformatic tools, such as the BLASTP program or Profile Hidden Markov models, to screen for and identify conserved protein sequences harboring a characteristic set of universally conserved functional domains typical of all terpene synthases. Of particular importance is the presence of variants of two universally conserved domains, the aspartate-rich DDXX(D/E) motif and the NSE/DTE triad, (N/D)DXX(S/T)XX(K/R)(D/E). Both domains have been implicated in the binding of the essential divalent cation, typically Mg2+, that is required for cyclization of the universal acyclic terpene precursors, such as farnesyl and geranyl diphosphate. The low level of overall sequence similarity among terpene synthases, however, has so far precluded any simple correlation of protein sequence with the structure of the cyclized terpene product. The actual biochemical function of a cryptic bacterial (or indeed any) terpene synthase must therefore be determined by direct experiment. Two common approaches are (i) incubation of the expressed recombinant protein with acyclic allylic diphosphate substrates and identification of the resultant terpene hydrocarbon or alcohol and (ii) in vivo expression in engineered bacterial hosts that can support the production of terpene metabolites. One of the most attractive features of the coordinated application of genome mining and biochemical characterization is that the discovery of natural products is directly coupled to the simultaneous discovery and exploitation of the responsible biosynthetic genes and enzymes. Bacterial genome mining has proved highly rewarding scientifically, already uncovering more than a dozen newly identified cyclic terpenes (many of them unique to bacteria) as well as several novel cyclization mechanisms. Moreover, bioinformatic analysis has identified more than 120 presumptive genes for bacterial terpene synthases that are now ripe for exploration. In this Account, we review a particularly rich vein we have mined in the genomes of two model Actinomycetes, Streptomyces coelicolor and Streptomyces avermitilis, from which the entire set of terpenoid biosynthetic genes and pathways have now been eluc...

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“…Together, these structures provide important clues regarding the mechanism of the unusual isoprenoid methylation reaction catalyzed by GPPMT. Although these are the first structures of an isoprenoid methyltransferase to be reported in the Protein Data Bank (www.rcsb.org), we note that the crystallization and preliminary X-ray crystallographic study of GPPMT from Streptomyces lasaliensis was recently reported (24). …”

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confidence: 95%

Structure of Geranyl Diphosphate C-Methyltransferase from Streptomyces coelicolor and Implications for the Mechanism of Isoprenoid Modification

et al. 2012

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Geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is the first methyltransferase discovered that modifies an acyclic isoprenoid diphosphate, geranyl diphosphate (GPP), to yield a non-canonical acyclic allylic diphosphate product, 2-methylgeranyl diphosphate, which serves as the substrate for a subsequent cyclization reaction catalyzed by a terpenoid cyclase, methylisoborneol synthase. Here, we report the crystal structures of GPPMT in complex with GPP or the substrate analogue geranyl-S-thiolodiphosphate (GSPP) along with S-adenosyl-l-homocysteine in the cofactor binding site, resulting from in situ demethylation of S-adenosyl-l-methionine, at 2.05 Å and 1.82 Å resolution, respectively. These structures suggest that both GPP and GSPP can undergo catalytic methylation in crystalline GPPMT, followed by dissociation of the isoprenoid product. S-adenosyl-l-homocysteine remains bound in the active site, however, and does not exchange with a fresh molecule of cofactor S-adenosyl-l-methionine. These structures provide important clues regarding the molecular mechanism of the reaction, especially with regard to the face of the 2,3 double bond of GPP that is methylated as well as the stabilization of the resulting carbocation intermediate through cation-π interactions.

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Crystallization and preliminary X-ray crystallographic study of a methyltransferase involved in 2-methylisoborneol biosynthesis inStreptomyces lasaliensis

Cited by 5 publications

References 12 publications

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependentC-methylation

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependentC-methylation

Exploration and Mining of the Bacterial Terpenome

Structure of Geranyl Diphosphate C-Methyltransferase from Streptomyces coelicolor and Implications for the Mechanism of Isoprenoid Modification

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