Biology subjects

Boal, A.

Publications and source records attributed to Boal, A..

3 recordsLinked to original sources

bioRxiv2026

Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity

The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached in an amide linkage to a target lysyl residue of a lipoyl carrier protein. This reaction is catalyzed by lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) superfamily. Lipoyl synthase requires two [4Fe-4S] clusters. One cluster is used to cleave SAM reductively to generate two 5'-deoxyadenosyl 5'-radicals (5'-dA*), which abstract the C6 and C8 hydrogen atoms (H*) of the substrate in two sequential steps. The second cluster, termed the auxiliary cluster, is consumed during turnover to provide the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX4CX5C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R306SS308Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in an essentially inactive protein due to poor substrate binding and positioning in the active site. Multiple substitutions of Ser308 were engineered. Most notable were the S308C and S308A variants, which greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl intermediate and the formation of a desaturated product, identified as a 6-octenoyl group. Furthermore, the 3Fe cluster formed during degradation of the auxiliary cluster during C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2, configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/727706v2_ufig2.gif" ALT="Figure 2"> View larger version (36K): org.highwire.dtl.DTLVardef@19e1f29org.highwire.dtl.DTLVardef@ff1e4org.highwire.dtl.DTLVardef@d1cf91org.highwire.dtl.DTLVardef@923d1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

A Substrate-triggered μ-Peroxodiiron(III) Intermediate in the 4-Choro-L-Lysine-Fragmenting Heme-Oxygenase-like Diiron Oxidase (HDO) BesC: Substrate Dissociation from, and C4 Targeting by, the Intermediate

The enzyme BesC from the {beta}-ethynyl-L-serine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-L-lysine (produced from L-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-L-allylglycine and can analogously fragment L-Lys itself. BesC belongs to the emerging family of O2-activating non-heme-diiron enzymes with the "heme-oxygenase-like" protein fold (HDOs). Here we show that binding of L-Lys or an analog triggers capture of O2 by the proteins diiron(II) cofactor to form a blue {micro}-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino-N-oxygenase domain of SznF. The [~] 5- and [~] 30-fold faster decay of the intermediate in reactions with 4-thia-L-Lys and (4RS)-chloro-DL-lysine than in the reaction with L-Lys itself, and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of L-allylglycine in the reaction with 4,4,5,5-[2H]-L-Lys, imply that the peroxide intermediate or a successor complex with which it reversibly interconverts initiates the oxidative fragmentation by abstracting hydrogen from C4. Surprisingly, the sluggish substrate L-Lys can dissociate after triggering the intermediate to form, thereby allowing one of the better substrates to bind and react. Observed linkage between Fe(II) and substrate binding suggests that the triggering event involves a previously documented (in SznF) ordering of the dynamic HDO architecture that contributes one of the iron sites, a hypothesis consistent with the observation that the diiron(III) product cluster produced upon decay of the intermediate spontaneously degrades, as it has been shown to do in all other HDOs studied to date.

biochemistry↗

bioRxiv2021

Structural basis for carbapenam C-alkylation by TokK, a B12-dependent radical SAM enzyme

Cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes acting during carbapenem antibiotic biosynthesis carry out radical-mediated methyl transfers that underlie the therapeutic usefulness of these essential medicines. Here we present x-ray crystal structures of TokK, which are representative of this functional class, containing its two metallocofactors and determined in the presence and absence of carbapenam substrate. The structures give the first visualization of a cobalamin-dependent radical SAM methylase that employs the radical mechanism shared by a vast majority of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggests that substrate positioning governs the rate of each methylation event. One Sentence SummaryStructural insight into a cobalamin-dependent radical SAM methylase that performs three sequential radical-mediated methylations to install the C6 side chain of a carbapenem antibiotic.

biochemistry↗