Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine {beta}-synthase

The catalytic potential for H₂S biogenesis and homocysteine clearance converge at the active site of cystathionine β-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes β-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H₂S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 Å) and an aminoacrylate intermediate (1.55 Å) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory “energy-sensing” CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.     

The 1',4'-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes

Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4'-aminopyrimidine aromatic rings, but only recently has participation of the 4'-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4'-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1',4'-iminopyrimidine and the 4'-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1',4'-iminopyrimidine form. The identification of the 1',4'-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis.     

Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases

In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e^- reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e^- reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.     

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.     

π-Frontier molecular orbitals in S = 2 ferryl species and elucidation of their contributions to reactivity

S = 2 Fe^IV═O species are key intermediates in the catalysis of most nonheme iron enzymes. This article presents detailed spectroscopic and high-level computational studies on a structurally-defined S = 2 Fe^IV═O species that define its frontier molecular orbitals, which allow its high reactivity. Importantly, there are both π- and σ-channels for reaction, and both are highly reactive because they develop dominant oxyl character at the transition state. These π- and σ-channels have different orientation dependences defining how the same substrate can undergo different reactions (H-atom abstraction vs. electrophilic aromatic attack) with Fe^IV═O sites in different enzymes, and how different substrates can undergo different reactions (hydroxylation vs. halogenation) with an Fe^IV═O species in the same enzyme.     

Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity

Thiamin (vitamin B₁) is an essential micronutrient needed as a cofactor for many central metabolic enzymes. Animals must have thiamin in their diet, whereas bacteria, fungi, and plants can biosynthesize it de novo from the condensation of a thiazole and a pyrimidine moiety. Although the routes to biosynthesize these two heterocycles are not conserved in different organisms, in all cases exogenous thiamin represses expression of one or more of the biosynthetic pathway genes. One important mechanism for this control is via thiamin-pyrophosphate (TPP) riboswitches, regions of the mRNA to which TPP can bind directly, thus facilitating fine-tuning to maintain homeostasis. However, there is little information on how modulation of riboswitches affects thiamin metabolism in vivo. Here we use the green alga, Chlamydomonas reinhardtii, which regulates both thiazole and pyrimidine biosynthesis with riboswitches in the THI4 (Thiamin 4) and THIC (Thiamin C) genes, respectively, to investigate this question. Our study reveals that regulation of thiamin metabolism is not the simple dogma of negative feedback control. Specifically, balancing the provision of both of the heterocycles of TPP appears to be an important requirement. Furthermore, we show that the Chlamydomonas THIC riboswitch is controlled by hydroxymethylpyrimidine pyrophosphate, as well as TPP, but with an identical alternative splicing mechanism. Similarly, the THI4 gene is responsive to thiazole. The study not only provides insight into the plasticity of the TPP riboswitches but also shows that their maintenance is likely to be a consequence of evolutionary need as a function of the organisms’ environment and the particular pathway used.     

A catalytic di-heme bis-Fe(IV) intermediate, alternative to an Fe(IV)=O porphyrin radical

High-valent iron species are powerful oxidizing agents in chemical and biological catalysis. The best characterized form of an Fe(V) equivalent described in biological systems is the combination of a b-type heme with Fe(IV)=O and a porphyrin or amino acid cation radical (termed Compound I). This work describes an alternative natural mechanism to store two oxidizing equivalents above the ferric state for biological oxidation reactions. MauG is an enzyme that utilizes two covalently bound c-type hemes to catalyze the biosynthesis of the protein-derived cofactor tryptophan tryptophylquinone. Its natural substrate is a monohydroxylated tryptophan residue present in a 119-kDa precursor protein. An EPR-silent di-heme reaction intermediate of MauG was trapped. Mössbauer spectroscopy revealed the presence of two distinct Fe(IV) species. One is consistent with an Fe(IV)=O (ferryl) species (δ = 0.06 mm/s, ΔE_Q = 1.70 mm/s). The other is assigned to an Fe(IV) heme species with two axial ligands from protein (δ = 0.17 mm/s, ΔE_Q = 2.54 mm/s), which has never before been described in nature. This bis-Fe(IV) intermediate is remarkably stable but readily reacts with its native substrate. These findings broaden our views of how proteins can stabilize a highly reactive oxidizing species and the scope of enzyme-catalyzed posttranslational modifications.     

Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase

Nitrogenase catalyzes the reduction of N₂ and protons to yield two NH₃ and one H₂. Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH₃), or hydrazine as substrate recently was shown to trap a common intermediate, designated I. It was further concluded that the two N-atoms of N₂ are hydrogenated alternately (“Alternating” (A) pathway). In the present work, Q-band CW EPR and ⁹⁵Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state “non-Kramers” doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of ^14,15N isotopologs plus ^1,2H isotopologs of methyldiazene. It is concluded that: H has NH₂ bound to FeMo-co and corresponds to the penultimate intermediate of N₂ hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH₃; I corresponds to the final intermediate in N₂ reduction, the state formed after accumulation of eight electrons/protons, with NH₃ still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the “prompt” alternating reaction pathway represents a draft mechanism for N₂ reduction by nitrogenase.     

A deoxyribozyme that harnesses light to repair thymine dimers in DNA

In vitro selection was used to investigate whether nucleic acid enzymes are capable of catalyzing photochemical reactions. The reaction chosen was photoreactivation of thymine cyclobutane dimers in DNA by using serotonin as cofactor and light of wavelengths longer than the absorption spectrum of DNA. Curiously, the dominant single-stranded DNA sequence selected, UV1A, was found to repair its internal thymine dimer substrate efficiently even in the absence of serotonin or any other cofactor. UV1C, a 42-nucleotide fragment of UV1A, repaired the thymine dimer substrate in trans (k_cat/k_uncat = 2.5 × 10⁴), showing optimal activity with 305 nm light and thus resembling naturally occurring photolyase enzymes. Mechanistic investigation of UV1C indicated that its catalytic role likely exceeded the mere positioning of the substrate in a conformation favorable for photoreactivation. A higher-order structure, likely a quadruplex, formed by specific guanine bases within the deoxyribozyme, was implicated as serving as a light-harvesting antenna, with photoreactivation of the thymine dimer proceeding possibly via electron donation from an excited guanine base. In a primordial “RNA world,” self-replicating nucleic acid populations may have been vulnerable to deactivation via UV light-mediated pyrimidine dimer formation. Photolyase nucleic acid enzymes such as the one described here could thus have played a role in preserving the integrity of such an RNA world.     

Uroporphyrinogen decarboxylation as a benchmark for the catalytic proficiency of enzymes

The magnitude of an enzyme's affinity for the altered substrate in the transition state exceeds its affinity for the substrate in the ground state by a factor matching the rate enhancement that the enzyme produces. Particularly remarkable are those enzymes that act as simple protein catalysts, without the assistance of metals or other cofactors. To determine the extent to which one such enzyme, human uroporphyrinogen decarboxylase, enhances the rate of substrate decarboxylation, we examined the rate of spontaneous decarboxylation of pyrrolyl-3-acetate. Extrapolation of first-order rate constants measured at elevated temperatures indicates that this reaction proceeds with a half-life of 2.3 × 10⁹ years at 25 °C in the absence of enzyme. This enzyme shows no significant homology with orotidine 5′-monophosphate decarboxylase (ODCase), another cofactorless enzyme that catalyzes a very slow reaction. It is proposed that, in both cases, a protonated basic residue (Arg-37 in the case of human UroD; Lys-93 in the case of yeast ODCase) furnishes a counterion that helps the scissile carboxylate group of the substrate leave water and enter a relatively nonpolar environment, stabilizes the incipient carbanion generated by the departure of CO₂, and supplies the proton that takes its place.     

Acute exercise increases resistance to oxidative stress in young but not older adults

A single bout of acute exercise increases oxidative stress and stimulates a transient increase in antioxidant enzymes. We asked whether this response would induce protection from a subsequent oxidative challenge, different from that of exercise, and whether the effects were affected by aging. We compared young (20 ± 1 years, n = 8) and older (58 ± 6 years, n = 9) healthy men and women. Resistance to oxidative stress was measured by the F₂-isoprostane response to forearm ischemia/reperfusion (I/R) trial. Each participant underwent the I/R trial twice, in random order; once after performing 45 min of cycling on the preceding day (IRX) and a control trial without any physical activity (IRC). Baseline F₂-isoprostane levels were significantly lower at IRX compared to IRC (P < 0.05) and not different between groups. F₂-isoprostane response to IRX was significantly lower compared to IRC in young (P < 0.05) but not different in the older group. Superoxide dismutase activity in response to acute exercise was significantly higher in young compared to older adults (P < 0.05). These data suggest that signal transduction of acute exercise may be impaired with aging. Repeated bouts of transient reactive oxygen species production as seen with regular exercise may be needed to increase resistance to oxidative stress in older individuals.     

Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface

The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70–DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1–70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual “tethered” binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein. Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422–433].     

Transition states of native and drug-resistant HIV-1 protease are the same

HIV-1 protease is an important target for the treatment of HIV/AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary ¹⁴C and ¹⁵N KIEs and secondary ³H and ¹⁸O KIEs for native and multidrug-resistant HIV-1 protease (I84V). We observed ¹⁴C KIEs (¹⁴V/K) of 1.029 ± 0.003 and 1.025 ± 0.005, ¹⁵N KIEs (¹⁵V/K) of 0.987 ± 0.004 and 0.989 ± 0.003, ¹⁸O KIEs (¹⁸V/K) of 0.999 ± 0.003 and 0.993 ± 0.003, and ³H KIEs (³V/K) KIEs of 0.968 ± 0.001 and 0.976 ± 0.001 for the native and I84V enzyme, respectively. The chemical reaction involves nucleophilic water attack at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.     

Structure-guided reprogramming of serine recombinase DNA sequence specificity

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences > 10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with > 80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.     

Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog

Cyclodipeptides are secondary metabolites biosynthesized by many bacteria and exhibit a wide array of biological activities. Recently, a new class of small proteins, named cyclodipeptide synthases (CDPS), which are unrelated to the typical nonribosomal peptide synthetases, was shown to generate several cyclodipeptides, using aminoacyl-tRNAs as substrates. The Mycobacterium tuberculosis CDPS, Rv2275, was found to generate cyclodityrosine through the formation of an aminoacyl-enzyme intermediate and to have a structure and oligomeric state similar to those of the class Ic aminoacyl-tRNA synthetases (aaRSs). However, the poor sequence conservation among CDPSs has raised questions about the architecture and catalytic mechanism of the identified homologs. Here we report the crystal structures of Bacillus licheniformis CDPS YvmC-Blic, in the apo form and complexed with substrate mimics, at 1.7–2.4-Å resolutions. The YvmC-Blic structure also exhibits similarity to the class Ic aaRSs catalytic domain. Our mutational analysis confirmed the importance of a set of residues for cyclodileucine formation among the conserved residues localized in the catalytic pocket. Our biochemical data indicated that YvmC-Blic binds tRNA and generates cyclodileucine as a monomer. We were also able to detect the presence of an aminoacyl-enzyme reaction intermediate, but not a dipeptide tRNA intermediate, whose existence was postulated for Rv2275. Instead, our results support a sequential catalytic mechanism for YvmC-Blic, with the successive attachment of two leucine residues on the enzyme via a conserved serine residue. Altogether, our findings suggest that all CDPS enzymes share a common aaRS-like architecture and a catalytic mechanism involving the formation of an enzyme-bound intermediate.     

Metal-organic charge transfer can produce biradical states and is mediated by conical intersections

The present paper illustrates key features of charge transfer between calcium atoms and prototype conjugated hydrocarbons (ethylene, benzene, and coronene) as elucidated by electronic structure calculations. One- and two-electron charge transfer is controlled by two sequential conical intersections. The two lowest electronic states that undergo a conical intersection have closed-shell and open-shell dominant configurations correlating with the 4s² and 4s¹3d¹ states of Ca, respectively. Unlike the neutral-ionic state crossing in, for example, hydrogen halides or alkali halides, the path from separated reactants to the conical intersection region is uphill and the charge-transferred state is a biradical. The lowest-energy adiabatic singlet state shows at least two minima along a single approach path of Ca to the π system: (i) a van der Waals complex with a doubly occupied highest molecular orbital, denoted , and a small negative charge on Ca and (ii) an open-shell singlet (biradical) at intermediate approach (Ca⋯C distance ≈2.5–2.7 Å) with molecular orbital structure ϕ₁ϕ₂, where ϕ₂ is an orbital showing significant charge transfer form Ca to the π-system, leading to a one-electron multicentered bond. A third minimum (iii) at shorter distances along the same path corresponding to a closed-shell state with molecular orbital structure has also been found; however, it does not necessarily represent the ground state at a given Ca⋯C distance in all three systems. The topography of the lowest adiabatic singlet potential energy surface is due to the one- and two-electron bonding patterns in Ca-π complexes.     

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH^-). The CPD repair mechanism involves electron transfer from photoexcited FADH^- to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH^-. The two electron transfer processes occur on time scales of 10^-10 and 10^-9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T^∘-) and electron return from T^∘- to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.     

Revealing the catalytic potential of an acyl-ACP desaturase: tandem selective oxidation of saturated fatty acids

It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of “unusual” e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. ¹⁸O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O₂-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.     

Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase

The scaffold protein for iron-sulfur cluster assembly, apo-IscU, populates two interconverting conformational states, one disordered (D) and one structured (S) as revealed by extensive NMR assignments. At pH 8 and 25 °C, approximately 70% of the protein is S, and the lifetimes of the states are 1.3 s (S) and 0.50 s (D). Zn(II) and Fe(II) each bind and stabilize structured (S-like) states. Single amino acid substitutions at conserved residues were found that shift the equilibrium toward either the S or the D state. Cluster assembly takes place in the complex between IscU and the cysteine desulfurase, IscS, and our NMR studies demonstrate that IscS binds preferentially the D form of apo-IscU. The addition of 10% IscS to IscU was found to greatly increase H/D exchange at protected amides of IscU, to increase the rate of the S → D reaction, and to decrease the rate of the D → S reaction. In the saturated IscU:IscS complex, IscU is largely disordered. In vitro cluster assembly reactions provided evidence for the functional importance of the S⇆D equilibrium. IscU variants that favor the S state were found to undergo a lag phase, not observed with the wild type, that delayed cluster assembly; variants that favor the D state were found to assemble less stable clusters at an intermediate rate without the lag. It appears that IscU has evolved to exist in a disordered conformational state that is the initial substrate for the desulfurase and to convert to a structured state that stabilizes the cluster once it is assembled.     

Mutagenesis of tryptophan199 suggests that hopping is required for MauG-dependent tryptophan tryptophylquinone biosynthesis

The diheme enzyme MauG catalyzes the posttranslational modification of the precursor protein of methylamine dehydrogenase (preMADH) to complete biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Catalysis proceeds through a high valent bis-Fe(IV) redox state and requires long-range electron transfer (ET), as the distance between the modified residues of preMADH and the nearest heme iron of MauG is 19.4 Å. Trp199 of MauG resides at the MauG-preMADH interface, positioned midway between the residues that are modified and the nearest heme. W199F and W199K mutations did not affect the spectroscopic and redox properties of MauG, or its ability to stabilize the bis-Fe(IV) state. Crystal structures of complexes of W199F/K MauG with preMADH showed no significant perturbation of the MauG-preMADH structure or protein interface. However, neither MauG variant was able to synthesize TTQ from preMADH. In contrast, an ET reaction from diferrous MauG to quinone MADH, which does not require the bis-Fe(IV) intermediate, was minimally affected by the W199F/K mutations. W199F/K MauGs were able to oxidize quinol MADH to form TTQ, the putative final two-electron oxidation of the biosynthetic process, but with k_cat/K_m values approximately 10% that of wild-type MauG. The differential effects of the W199F/K mutations on these three different reactions are explained by a critical role for Trp199 in mediating multistep hopping from preMADH to bis-Fe(IV) MauG during the long-range ET that is required for TTQ biosynthesis.