The ß-latch structural element of the SufS cysteine desulfurase mediates active site accessibility and SufE transpersulfurase positioning.

Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-dependent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is protected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "ß-latch" structural element that includes the α6 helix, a glycine-rich loop, a ß-hairpin, and a cis-proline residue. To identify a functional role for the ß-latch, we used site-directed mutagenesis to obtain the N99D and N99A SufS variants. N99 is a conserved residue that connects the α6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a distorted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high-affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demonstrate that the ß-latch does not affect the chemistry of persulfide formation but does protect it from undesired reductants. The data also indicate the ß-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.

Biochemical characterization of 2-phosphinomethylmalate synthase from Streptomyces hygroscopicus: A member of the DRE-TIM metallolyase superfamily.

2-Phosphinomethylmalate synthase (PMMS) from Streptomyces hygroscopicus catalyzes the first step in the biosynthesis of the herbicide bialophos using 3-phosphinopyruvic acid and acetyl coenzyme A as substrates to form 2-phosphinomethylmalic acid and coenzyme A. PMMS belongs to the Claisen condensation-like (CC-like) subgroup of the DRE-TIM metallolyase superfamily, which uses conserved active site architecture to catalyze a functionally-diverse set of reactions. Analysis of a sequence similarity network for the CC-like subgroup identified PMMS and the related R-citrate synthase in an early-diverging cluster suggesting that this group of sequences are more distinct in relation to other Claisen-condensation subgroup members. To better understand the structure/function landscape of the CC-like subgroup PMMS was recombinantly expressed in Escherichia coli, purified, and characterized with respect to its enzymatic properties. Using oxaloacetate as a substrate analog, the recombinantly-produced enzyme exhibited improved Michaelis constants relative to the previously reported natively-produced enzyme. Results from pH rate profiles and kinetic isotope effects were consistent with results from other members of the CC-like subgroup supporting acid-base chemistry and hydrolysis of the direct Claisen-condensation product as the rate-determining step. Results of site-directed mutagenesis experiments indicate that PMMS uses an active-site architecture similar to homocitrate synthase to select for a dicarboxylic acid substrate.