Identification and characterization of Helicobacter pylori O‐acetylserine‐dependent cystathionine β‐synthase, a distinct member of the PLP‐II family

Abstract

O-acetylserine sulfhydrylase (OASS) and cystathionine β-synthase (CBS) are members of the PLP-II family, and involved in L-cysteine production. OASS produces L-cysteine via a de novo pathway while CBS participates in the reverse transsulfuration pathway. O-acetylserine-dependent CBS (OCBS) was previously identified as a new member of the PLP-II family, which are predominantly seen in bacteria. The bacterium Helicobacter pylori possess only one OASS (hp0107) gene and we showed that the protein coded by this gene actually functions as an OCBS and utilizes L-homocysteine and O-acetylserine (OAS) to produce cystathionine. HpOCBS did not show CBS activity with the substrate L-serine and required OAS exclusively. The HpOCBS structure in complex with methionine showed a closed cleft state, explaining the initial mode of substrate binding. Sequence and structural analyses showed differences between the active sites of OCBS and CBS, and explain their different substrate preferences. We identified three hydrophobic residues near the active site of OCBS, corresponding to one serine and two tyrosine residues in CBSs. Mutational studies were performed on HpOCBS and Saccharomyces cerevisiae CBS. A ScCBS double mutant (Y158F/Y226V) did not display activity with L-serine, indicating indispensability of these polar residues for selecting substrate L-serine, however, did show activity with OAS.

Cystathionine β-synthase is involved in cysteine biosynthesis and H2S generation in Toxoplasma gondii

“…Similar to CBSs, the catalytic site of lanthionine synthase includes five conserved loops/blocks configuring the catalytic site. Loop 1, also known as the “asparagine loop” 36 , is known to interact with the first substrate involved in the β-replacement reaction, e.g. l -OAS 37 or l -Ser 5 , and also helps stabilize the aminoacrylate intermediate; Loop-1 contains a conserved serine (S82 in yeast CBS 26 , S84 in TgCBS) that has been postulated to interact with the second substrate, l -Hcys during the canonical β-replacement 26 , 36 .…”

“…Loop 4 and Loop 5 contribute to stabilize the orientation of PLP through interactions with the pyridine ring. ( b ) Partial sequence alignment of loop-3 in Toxoplasma gondii cystathionine β-synthase (TgCBS); human cystathionine β-synthase (HsCBS); Fusobacterium nucleatum lanthionine synthase (FnLS) 35 ; Helicobacter pylori O -acetylserine-dependent CBS (HpOCBS) 36 . …”

Structure of Cystathionine β-Synthase from Toxoplasma gondii, a key enzyme in its H2S production machinery

“…4A) (27), (43) and contains four out of the five consensus sequences that participate in the interaction with the different CBS substrates (blocks B2, B3, B4 and B5 in Fig. 4A) (41). The main differences with the human protein are in the region comprising residues 232-238 of block B3 (Fig.…”

“…In particular, TgCBS can use both Ser and O-acetylserine (OAS) to produce Cth (24). This catalytic ability links TgCBS to other enzymes such as OCBSs (41) and OASS, which also employ OAS as a substrate. Besides a role in cysteine biosynthesis, TgCBS is also strongly implicated in the production of H2S, being highly efficient in catalyzing the β-replacement of Cys with HCys (24).…”

Brucella oviscysteine biosynthesis contributes to peroxide stress survival and fitness in the intracellular niche

“…It is possible that CysK1 and CysK2 do not catalyze the same reaction, but rather determine cysteine biosynthesis through two distinct routes. A recent report of such a case is the cystathionine b-synthase of Helicobacter pylori, which retains some O-acetylserine sulfhydrylase activity (33); this enzyme shares some features of primary structure with B. ovis CysK2. CysKfamily enzymes can also have functions beyond direct involvement in cysteine metabolism (34), which may influence interpretation of our results.…”