Folding and activity of mutant cystathionine β-synthase depends on the position and nature of the purification tag: Characterization of the R266K CBS mutant

Abstract

Cystathionine beta-synthase (CBS), a heme-containing PLP-dependent enzyme, catalyzes the condensation of serine and homocysteine to yield cystathionine. Missense mutations in CBS, the most common cause of homocystinuria, often result in misfolded proteins. Arginine 266, where the pathogenic missense mutation R266K was identified, appears to be involved in the communication between heme and the PLP-containing catalytic center. Here, we assessed the effect of a short affinity tag (6xHis) compared to a bulky fusion partner (glutathione S-transferase - GST) on CBS wild type (WT) and R266K mutant enzyme properties. While WT CBS was successfully expressed either in conjunction with a GST or with a 6xHis tag, the mutant R266K CBS had no activity, did not form native tetramers and did not respond to chemical chaperone treatment when expressed with a GST fusion partner. Interestingly, expression of R266K CBS constructs with a 6xHis tag at either end yielded active enzymes. The purified, predominantly tetrameric, R266K CBS with a C-terminal 6xHis tag had ~ 82% of the activity of a corresponding WT CBS construct. Results from thermal pre-treatment of the enzyme and the denaturation profile of R266K suggests a lower thermal stability of the mutant enzyme compared to WT, presumably due to a disturbed heme environment.

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S -adenosylmethionine

“…The corresponding mutagenesis oligonucleotides were as follows (capital letters designate the codon of the mutated residue): 5′-ccgcttcgacagtccgTCAtcccatgtgggtgtt (FWD E201S), 5′-aacacccacatgggaTGAcggactgtcgaagcgg (REV E201S), 5′-gaatgcccgcttcGTCagtccggaatccc (FWD D198V), 5′-gggattccggactGACgaagcgggcattc (REV D198V), 5′-cctgggtaacatgctgCTAtcctgctggcgggca (FWD S466L), and 5′-tgcccgccagcagggaTAGcagcatgttacccagg (REV S466L). Purification of recombinant proteins followed the protocols that we developed for various hCBS constructs carrying either cleavable GST at the N terminus or a permanent 6xHis tag at the C terminus with a few modifications (11,31,33). Protein concentration was determined by the Bradford method (Thermo Pierce) using BSA as a standard according to the manufacturer's recommendations.…”

“…Protein concentration was determined by the Bradford method (Thermo Pierce) using BSA as a standard according to the manufacturer's recommendations. The CBS activity in the classical reaction was determined by a previously described radioisotope assay using [ 14 C(U)] L-serine as the labeled substrate, essentially as described elsewhere (11,33,34).…”

“…The central catalytic core shows the fold of the type II family PLP-dependent enzymes (5,6). Finally, the C-terminal region consists of a tandem pair of CBS motifs (7)(8)(9) that bind S-adenosylmethionine (AdoMet) and lead to an increase in catalytic activity by up to fivefold (10,11). The CBS motif pair, commonly known as a "Bateman module" (12,13), is responsible for CBS subunit tetramerization (14,15).…”

Structural basis of regulation and oligomerization of human cystathionine β-synthase, the central enzyme of transsulfuration

“…Preparation of the recombinant modified hCBSOPTΔ516-525, C-hCBSOPTΔ516-525, C-hCBSOPTΔ516-525 D444N, and C-hCBSOPTΔ1-39Δ516-525 D444N enzymes followed the protocols that we developed for various hCBS constructs either expressed with cleavable GST at their N terminus (37) or carrying a permanent 6xHis tag at their C terminus (38), with a few modifications (22). The deletions and the D444N pathogenic mutation were introduced by using a QuikChangeII XL mutagenesis kit (Agilent) according to the manufacturer's recommendations.…”

“…The deletions and the D444N pathogenic mutation were introduced by using a QuikChangeII XL mutagenesis kit (Agilent) according to the manufacturer's recommendations. Denaturing and native protein gel electrophoresis, Western blot, and the CBS activity assay in the absence and presence of AdoMet were performed essentially as described previously (37,38). The crystals were grown by the sitting and/or hanging-drop vapor-diffusion method at 293 K in 96-well and 24-well crystallization plates, respectively, according to the protocol described previously (22).…”