Biotin Synthase Exhibits Burst Kinetics and Multiple Turnovers in the Absence of Inhibition by Products and Product-Related Biomolecules

Abstract

Biotin synthase (BS) is a member of the "SAM radical" superfamily of enzymes, which catalyze reactions in which the reversible or irreversible oxidation of various substrates is coupled to the reduction of the S-adenosyl-l-methionine (AdoMet) sulfonium to generate methionine and 5'-deoxyadenosine (dAH). Prior studies have demonstrated that these products are modest inhibitors of BS and other members of this enzyme family. In addition, the in vivo catalytic activity of Escherichia coli BS requires expression of 5'-methylthioadenosine/S-adenosyl-l-homocysteine nucleosidase, which hydrolyzes 5'-methylthioadenosine (MTA), S-adenosyl-l-homocysteine (AdoHcy), and dAH. In the present work, we confirm that dAH is a modest inhibitor of BS (K(i) = 20 μM) and show that cooperative binding of dAH with excess methionine results in a 3-fold enhancement of this inhibition. However, with regard to the other substrates of MTA/AdoHcy nucleosidase, we demonstrate that AdoHcy is a potent inhibitor of BS (K(i) ≤ 650 nM) while MTA is not an inhibitor. Inhibition by both dAH and AdoHcy likely accounts for the in vivo requirement for MTA/AdoHcy nucleosidase and may help to explain some of the experimental disparities between various laboratories studying BS. In addition, we examine possible inhibition by other AdoMet-related biomolecules present as common contaminants in commercial AdoMet preparations and/or generated during an assay, as well as by sinefungin, a natural product that is a known inhibitor of several AdoMet-dependent enzymes. Finally, we examine the catalytic activity of BS with highly purified AdoMet in the presence of MTAN to relieve product inhibition and present evidence suggesting that the enzyme is half-site active and capable of undergoing multiple turnovers in vitro.

Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis

“…Assuming the substrate/enzyme molar ratio used in our in vitro experiments to be saturating for the enzyme, the k cat value for the ester hydrolysis by BioH can be minimally estimated to be 60 s −1 . This value is orders of magnitude greater than those reported for the downstream enzymes of the pathway (20)(21)(22)(23). This mismatch of BioH catalytic activity with the other enzymes of the pathway and with the physiological need for only extremely modest amounts of biotin may indicate that the enzyme has not yet been fully integrated into E. coli biotin synthesis.…”

“…1), may be an intrinsically poor catalyst (4). This lack of catalytic efficiency is also characteristic of the downstream enzymes of the pathway (20)(21)(22)(23), and has been attributed to the traces of biotin required for cellular growth (24). To verify this premise, we sought to determine the kinetic constants for the esterase reaction mediated by BioH and attempted several in vitro assays, all of which were unsuccessful.…”

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…In the presence of iron sulfur cluster biosynthetic machinery that can reassemble the [2Fe-2S] cluster after each reaction cycle, catalytic activity of BioB has been demonstrated [59]. It may be that a similar reassembly process is required for the [4Fe-4S] auxiliary cluster of lipoyl synthase, but this remains experimentally untested.…”

The BioC O-Methyltransferase Catalyzes Methyl Esterification of Malonyl-Acyl Carrier Protein, an Essential Step in Biotin Synthesis

“…SAM is a highly unstable molecule (17,19). The biologically active (S,S) configuration of SAM is prone to spontaneous racemization at the sulfonium center to yield the inactive (R,S)-diastereomer (19).…”

“…SAM can also spontaneously degrade by intramolecular S N 2 attack of the methionine carboxylate on the ␥-carbon into methylthioadenosine and homocysteine (19). Typically commercial SAM is extracted from yeast and purified by HPLC to give preparations containing only ϳ43% of (S,S)-SAM, the biologically active species, with the remainder composed of the racemized species, degraded products such as S-adenosylhomocysteine (SAH) and as much as 10% of unidentified substances (17). These contaminants are known to interfere with SAM-dependent enzymes and are difficult to remove (17).…”