Cystathionine β-synthase (CBS)2
(L-Serine hydrolyase (adding homocysteine), EC 18.104.22.168) is central to sulfur amino acid metabolism in eukaryotes, and complete CBS deficiency is the principal cause of homocystinuria in humans. The CBS-catalyzed condensation of serine and homocysteine generates cystathionine which is then cleaved with cystathionine γ-lyase (CGL) to yield cysteine [1
Cysteine synthase (O
-acetylserine sulfhydrase, OASS; CS), the CBS counterpart in prokaryotes, plants and enteric protozoa, catalyzes the formation of cysteine from O
-acetylserine and hydrogen sulfide (H2
S) with the concomitant release of acetic acid. Unlike mammals, these organisms are able to assimilate inorganic sulfur into organic sulfur and are therefore essential players in the sulfur cycle in nature. CS and CBS are affiliated with the large β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes and have a high degree of sequence and structural conservation within the catalytic core [2
], (see also www.uchsc.edu/sm/cbs/cbsdata/cbsprotein.htm
). An evolutionary relationship between these enzymes is also illustrated by the fact that some CBS enzymes have retained CS activity [4
]. CS and CBS, together with other enzymes such as β-cyanoalanine synthase, the β-subunit of tryptophan synthase and cysteine lyase, catalyze primarily β-replacement reactions [5
]. Despite catalyzing mostly one reaction type, CS and CBS can utilize a wide range of substrates [7
CBS is the only PLP dependent enzyme which contains heme [7
]. The heme is co-ordinated by Cys 52 and His 65 residues located in the N-terminal region of the enzyme. 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
], thus contradicting previous reports that have shown catalytic involvement [12
]. CBS from lower eukaryotes such as Saccharomyces cerevisiae
do not contain heme [8
]. 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.
Human CBS activity is regulated by S
-adenosyl-L-methionine (AdoMet) [14
]. Interestingly, all CS and some CBS enzymes from lower eukaryotes are not responsive to AdoMet suggesting that the requirement by CBS of a heme moiety and regulation by AdoMet may have emerged at the same time evolutionarily and may be interrelated.
The purification of CBS from mammalian tissues is complicated by its tendency for aggregation and its susceptibility to proteolysis [15
]. Previously, an Escherichia coli
expression system using β-galactosidase (β-gal) was used for the purification of CBS [16
]. This system had several disadvantages. There was variable undesirable proteolytic cleavage within E. coli
between β-gal and CBS. The proteolytic cleavage produced free β-gal and CBS prematurely making it impossible to use an affinity chromatography step to obtain purified CBS. Because of these complications, we decided to purify CBS using an alternate expression system. The expression of CBS with a Glutathione-S
-Transferase (GST) affinity tag was first described by Warren Kruger’s laboratory [17
]. The disadvantage of that system was that the cleaved purified CBS still retained 11 non-CBS residues at its N-terminus [18
].We have previously expressed human CBS with a GST tag, which upon purification and removal of the fusion partner yielded CBS with 23 extra residues at the N-terminus [19
]. Subsequently, we demonstrated that N-terminal elongation altered the affinity of the enzyme for Hcy. This enzyme had ~9-fold lower Km
for Hcy [20
] compared to the WT enzyme [21
] and was used to obtain the first CBS structure [22
]. Here we describe a method to purify CBS using a GST fusion protein which yields CBS as close to normal sequence as possible. Using our construct we are able to obtain a purified enzyme with only one extra small amino acid residue, glycine, at the Nterminus of the purified CBS protein.
The new method gives us several other benefits. First, the vector we chose has a long recognition sequence for the precision protease cleavage site which provides a convenient long hinge region between GST and CBS allowing independent folding of the two enzymes. Second, this system gives the added benefit of a shorter fusion partner which enables us to rapidly purify large amounts of CBS in an easy, two-step procedure. In this paper we describe the expression and purification of the full-length wild type CBS as well as CBS which does not contain the C-terminal regulatory region (1–413) and CBS lacking both the N- and C-terminal regions (71–400). The 71–400 CBS does not contain heme and is highly conserved with the CS family of enzymes. We have further characterized all three forms of CBS with respect to their biochemical and physical properties.