Yeast Cystathionine β-Synthase Is a Pyridoxal Phosphate Enzyme but, Unlike the Human Enzyme, Is Not a Heme Protein

Abstract

Our studies of cystathionine ␤-synthase from Saccharomyces cerevisiae (yeast) are aimed at (1) clarifying the cofactor dependence and catalytic mechanism and (2) obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine ␤-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine ␤-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine ␤-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.Elevated plasma homocysteine is an important risk factor in coronary heart disease and other human diseases (1-3). One of the two major routes for detoxication of homocysteine is the pyridoxal phosphate (PLP) 1 -dependent ␤-replacement reactionThe deduced sequences of human (4,5), rat (6), and Saccharomyces cerevisiae (yeast) (7,8) CBS are similar. The finding that human CBS complements the cysteine auxotrophy of a yeast strain lacking CBS (5) demonstrates the functional conservation of the human and yeast genes.The remarkable observation that the sequence of rat CBS (6) is identical to the sequence of rat hemoprotein H-450 (9) led to the discovery that rat and human CBS contain both PLP and heme (10). Heme may play a role in redox regulation of the human enzyme and in binding homocysteine (11,12). Although yeast CBS has been purified to homogeneity (13), the absorption spectrum and cofactor content have not been reported. Here, we demonstrate that purified yeast CBS contains PLP but not heme. Because the absence of heme facilitates spectroscopic studies of the PLP and of enzyme-substrate intermediates, we are able to demonstrate directly that CBS converts L-serine to an aminoacrylate intermediate, as expected for a PLP enzyme that catalyzes a ␤-replacement reaction (14,15). EXPERIMENTAL PROCEDURESChemicals-L-Cystathionine and L-serine were from Fluka. ␦-Aminolevulinic acid, L-homocysteine thiolactone, aprotinin, pepstatin A, leupeptin, benzamidine hydrochloride, TPCK, TLCK, and PMSF were from S...

Characterization of Transsulfuration and Cysteine Biosynthetic Pathways in the Protozoan Hemoflagellate, Trypanosoma cruzi

“…The absorption spectrum of rTcCBS exhibited major peaks at 280 and 410 nm in a ratio of 1:0.16, typical of a pyridoxal phosphate-containing enzyme (43). However, no peak was visible around 428 nm, which was indicative of heme in case of rat and human CBS (43). Removal of the pyridoxal phosphate resulted in an apoenzyme having no absorbance in the visible range (data not shown).…”

“…Unlike human CBS, rTc-CBS was not activated by the presence of 1 mM S-adenosylmethionine and did not contain detectable amounts of heme (data not shown). The absorption spectrum of rTcCBS exhibited major peaks at 280 and 410 nm in a ratio of 1:0.16, typical of a pyridoxal phosphate-containing enzyme (43). However, no peak was visible around 428 nm, which was indicative of heme in case of rat and human CBS (43).…”

Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

“…The function of this ligand is yet to be determined; however, several laboratories have shown that it is not directly involved in the catalytic process [8][9][10][11], thus contradicting previous reports that have shown catalytic involvement [12,13]. CBS from lower eukaryotes such as Saccharomyces cerevisiae do not contain heme [8,9]. Thus the absence of this cofactor in CBS from lower eukaryotes suggests that the function of heme in this enzyme is unique to higher organisms.…”

Identification and functional analysis of cystathionine beta-synthase gene mutations in patients with homocystinuria

“…In vitro enzyme reaction and TLC The CBS enzyme activity was determined by modification of previously reported methods (Jhee et al 2000;Shan and Kruger 1998). A total of 50 ll of reaction volume contained 5 ll of 1-M Tris-HCl (pH 8.6), 2.5 ll of 5-mM PLP, 2.5 ll of 10 mg/dl BSA, 5 ll of 10-mM cystathionine, 1 ll of 14 C-serine, 1 ll of 50-mM AdoMet, 165 lg of calibrated CBS protein for the NIH3T3 cell line, and 80 lg of calibrated CBS protein for the COS7 cell line.…”