Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT fromSynechocystissp. PCC 6803

Abstract

Malonyl-coenzymeA:acyl-carrier protein transacylase (MCAT), which catalyzes the transfer of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP), is an essential enzyme in type II fatty-acid synthesis. The enzyme MCAT from Synechocystis sp. PCC 6803 (spMCAT), the first MCAT counterpart from a cyanobacterium, was cloned, purified and crystallized in order to determine its three-dimensional crystal structure. A higher-quality crystal with better diffraction was obtained by crystallization optimization. The crystal diffracted to 1.8 Å resolution and belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 43.22, b = 149.21, c = 40.59 Å . Matthews coefficient calculations indicated that the crystal contained one spMCAT molecule in the asymmetric unit with a Matthews coefficient of 2.18 Å 3 Da À1 and a solvent content of 43.65%.

Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering

“…PCC 6803 was solved, showing a 40% sequence identity with FabD from E. coli . 79 Also, MCAT from S. aureus and Streptococcus pneumoniae were recently described, suggesting subtle differences in substrate specificity. 80 Interestingly, the identification and characterization of a type II homologous MCAT in human mitochondria helped decipher the presence of a type II FAS in a type I FAS utilizing organism.…”

Engineering the fatty acid synthesis pathway in Synechococcus elongatus PCC 7942 improves omega-3 fatty acid production

“…The enzymes involved in the FA synthesis pathway display high amino acid similarity between cyanobacterial and E. coli homologs [ 17 , 18 ]. In fact, individual subunits of the Ss7002 and E. coli FAS complexes were qualitatively interchangeable in vitro, using purified protein components [ 17 ].…”

Structure‐directed construction of a high‐performance version of the enzyme FabG from the photosynthetic microorganismSynechocystis sp. PCC 6803

“…The lysate was centrifuged for 30 min at 12 000× g at 4 °C. The supernatant containing the soluble SpFabG and SpPhaB proteins was then added to Ni–NTA resin (Qiagen) as described by Liu et al[20], and the eluted proteins were further purified on a superdex 200 column by ÄKTA prime plus (GE) equilibrated with Tris–HCl buffer (50 mM Tris–HCl pH 7.8, 300 mM NaCl, 1 mM EDTA, 5% glycerol (v/v), and 2 mM β‐mercaptoethanol). The fractions containing SpFabG and SpPhaB were concentrated using an Amicon concentrator 10kDa (Millipore), and the protein concentrations were determined using the Bradford assay with BSA as the standard.…”