The Arabidopsis thaliana ortholog of a purported maize cholinesterase gene encodes a GDSL-lipase

Abstract

Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family.

Fig. 1

Phylogenetic analysis of homologs of the maize gene encoding for hypothetical protein LOC606473 (‘ache’). Analysis of the first 98 hits of a blastp search was performed on the Phylogeny.fr platform by aligning the sequences with MUSCLE (v3.7), reconstructing the phylogenetic tree with the BioNJ program, and drawing the circular phylogram with TreeDyn (v198.3) as detailed in the “Materials and Methods” section. Five clusters of orthologs can be distinguished with the first three more closely grouped: A1 (red), A2 (purple), A3 (green), B (magenta), and C (blue). All three major clusters included monocot (diamonds) and eudicot (circles) members. Numbers refer to the following accessions: 1, Z. mays (maize, NP_001105800); 2, S. bicolor (sorghum, XP_002463099); 3, O. sativa (rice, NP_001060129); 4, V. vinifera (grape, XP_002282372); 5, A. thaliana (NP_189274); 6, P. trichocarpa (poplar, XP_002314590); 7, R. communis (castor bean, XP_002530043); 8, H. brasiliensis (Para rubber, Q7Y1X1); 9, D. carota (carrot, BAF80349); 10, M. sativa (alfalfa,AAB41547); 11, G. max (soybean, ACU20252); 12, S. europaea (BAI23204); 13, Macroptilium atropurpureum (siratro, BAG09557). Insert: Homology of accessions constituting the five clusters to the maize ‘ache’ gene. Ranges of percent identity (I) and similarity (S) as determined by blastp are shown

Fig. 2. Accessions belonging to clusters A1-A3 are highly homologous to each other. The thirteen accessions indicated in Fig. 1 were aligned using the T-Coffee program, and the degree of similarity is shown as a heat diagram superimposed on the sequences

Fig. 3

Comparison of the intron–exon architecture of orthologs of the maize ‘ache’ gene, Intron, exon, 5′-UTR and 3′-UTR lengths were deduced by comparing genomic sequences to those of the mRNA sequences. Four cluster A sequences from maize, sorghum, rice and A. thaliana are shown. See “Materials and methods” for the accession numbers of these and additional sequences whose gene structure was analyzed. UTRs—blue, exons—red, introns—thin black lines

Fig. 4

E. coli –expressed At3g26430 protein cannot hydrolyze ACh. Expression of At3g26430 was induced by 0.3 mM IPTG for 2 h. Cells were homogenized by sonication and proteins were separated by centrifugation into soluble and insoluble fractions. Control E. coli cells harboring a non-related plasmid were similarly treated. The potential of protein(s) in the soluble fractions obtained from At3g26430- and control cell extracts to hydrolyze AtCh was assayed (see “Materials and Methods”). As a positive control, the soluble fractions were “spiked” with purified plant-derived human BChE and AtCh hydrolyzing activity similarly assayed. Insert: Proteins were extracted from At3g26430-E. coli cells prior to induction (lane 1), or following induction (lane 2), and then separated into soluble (lane 3) or insoluble (lane 4) fractions, resolved by SDS-PAGE and subjected to immunoblot analysis using anti-His tag Abs. The arrow points to the band corresponding to the At3g26430 protein

Fig. 5

Esterase and lipase activities of At3g26430 over-expressed in A. thaliana. Over expression of At3g26430 in transgenic A. thaliana plants as compared to WT plants was determined using semi quantitative PCR with actin serving as a housekeeping gene control (Insert). ChE, general esterase and lipase activities were assayed and results are expressed as percent of the activity levels in WT plants (dashed horizontal line). Results reflect at least three repeats (mean ± SEM)

Fig. 6

Histochemical staining to detect cholinesterase activity. Tissue distribution of ChE activity was determined by the Karnovsky and Roots in situ activity staining procedure. 9-day old seedlings of WT plants, and plants that overexpress At3g26430 or human AChE (Muralidharan and Mor, unpublished and Mor and Soreq 2004) were incubated in solutions containing the substrate ATCh (+) or solutions lacking the substrate (−). Rust-colored cupric ferrocyanide precipitate is deposited following the reduction of ferricyanide by the ChE-generated thiocholine. Only plants that express the bona fide ChE, human AChE, showed significant staining in the presence of the substrate, whereas only very slight staining was visible in WT and At3g26430 plants

Fig. 7

At3g26430 is likely a GDS(L) lipases belonging to the SGNH hydrolase clan. True cholinesterases (a, represented by human AChE) bear no homology to the At3g26430 protein (b, drawn to scale with a) with which they share only minimal superficial similarity protein in the form of analogous catalytic triads (blue dots). In contrast the At3g26430 protein is highly homologous to the members of the SGNH hydrolase clan and specifically to members of the GDS(L) lipase family (b, namesake residues are marked by asterisks). Particularly well-conserved are the four SGNH blocks that characterize the clan, which is represent here by several esterases belonging to the “seed” set of sequences for the GDS(L) lipase family (c, Pfam PF00657). The degree of sequence homology in these conserved blocks is shown as a heat diagram superimposed on the sequences. The UniProt accession numbers of the sequences compared to At3g26430 (A. thaliana) are: Q07792 (Vibrio mimicus); P0ADA1 (Escherichia coli), Q97DM5 (Clostridium acetobutylicum); P41734 (Saccharomyces cerevisiae); P28039 (Homo sapiens); Q08ET5 (Capsicum annuum). The latter is not on the Pfam seed list but was recently shown to be a lipase (Hong et al. 2008)

Fig. 8

Enzyme kinetics of At3g26430's serine hydrolase activity. At3g26430 (overexpressed in E. coli) was assayed for its ability to hydrolyze the lipase substrates p-nitrophenyl acetate (PNPA) (a), p-nitrophenyl butyrate (PNPB) (b) and p-nitrophenyl palmitate (PNPP). Reaction velocities were measured as a function of substrate concentration and the Michaelis constant (K_M) was calculated by non-linear regression using the Prism software. Inserts: Lineweaver–Burk analysis. PNPA hydrolysis was greatly inhibited. (d) PNPA hydrolysis was inhibited by PMSF (broken line), but not by NB (dotted line)