Function and Structure of MalA/MalA', Iterative Halogenases for Late-Stage C-H Functionalization of Indole Alkaloids.

Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.

Alternating access in maltose transporter mediated by rigid-body rotations.

ATP-binding cassette transporters couple ATP hydrolysis to substrate translocation through an alternating access mechanism, but the nature of the conformational changes in a transport cycle remains elusive. Previously we reported the structure of the maltose transporter MalFGK(2) in an outward-facing conformation in which the transmembrane (TM) helices outline a substrate-binding pocket open toward the periplasmic surface and ATP is poised for hydrolysis along the closed nucleotide-binding dimer interface. Here we report the structure of the nucleotide-free maltose transporter in which the substrate binding pocket is only accessible from the cytoplasm and the nucleotide-binding interface is open. Comparison of the same transporter crystallized in two different conformations reveals that alternating access involves rigid-body rotations of the TM subdomains that are coupled to the closure and opening of the nucleotide-binding domain interface. The comparison also reveals that point mutations enabling binding protein-independent transport line dynamic interfaces in the TM region.

Crystal structure of a catalytic intermediate of the maltose transporter.

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-A crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP.

Conformational switch triggered by alpha-ketoglutarate in a halogenase of curacin A biosynthesis.

The CurA halogenase (Hal) catalyzes a cryptic chlorination leading to cyclopropane ring formation in the synthesis of the natural product curacin A. Hal belongs to a family of enzymes that use Fe(2+), O(2) and alpha-ketoglutarate (alphaKG) to perform a variety of halogenation reactions in natural product biosynthesis. Crystal structures of the enzyme in five ligand states reveal strikingly different open and closed conformations dependent on alphaKG binding. The open form represents ligand-free enzyme, preventing substrate from entering the active site until both alphaKG and chloride are bound, while the closed form represents the holoenzyme with alphaKG and chloride coordinated to iron. Candidate amino acid residues involved in substrate recognition were identified by site-directed mutagenesis. These new structures provide direct evidence of a conformational switch driven by alphaKG leading to chlorination of an early pathway intermediate.

Insights from the sea: structural biology of marine polyketide synthases.

The world's oceans are a rich source of natural products with extremely interesting chemistry. Biosynthetic pathways have been worked out for a few, and the story is being enriched with crystal structures of interesting pathway enzymes. By far, the greatest number of structural insights from marine biosynthetic pathways has originated with studies of curacin A, a poster child for interesting marine chemistry with its cyclopropane and thiazoline rings, internal cis double bond, and terminal alkene. Using the curacin A pathway as a model, structural details are now available for a novel loading enzyme with remarkable dual decarboxylase and acetyltransferase activities, an Fe(2+)/α-ketoglutarate-dependent halogenase that dictates substrate binding order through conformational changes, a decarboxylase that establishes regiochemistry for cyclopropane formation, and a thioesterase with specificity for ß-sulfated substrates that lead to terminal alkene offloading. The four curacin A pathway dehydratases reveal an intrinsic flexibility that may accommodate bulky or stiff polyketide intermediates. In the salinosporamide A pathway, active site volume determines the halide specificity of a halogenase that catalyzes for the synthesis of a halogenated building block. Structures of a number of putative polyketide cyclases may help in understanding reaction mechanisms and substrate specificities although their substrates are presently unknown.

Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB.

The KorB protein of the broad-host-range plasmid RP4 acts as a multifunctional regulator of plasmid housekeeping genes, including those responsible for replication, maintenance and conjugation. Additionally, KorB functions as the ParB analog of the plasmid's partitioning system. The protein structure consists of eight helices, two of which belong to a predicted helix-turn-helix motif. Each half-site of the palindromic operator DNA binds one copy of the protein in the major groove. As confirmed by mutagenesis, recognition specificity is based mainly on two side chain interactions outside the helix-turn-helix motif with two bases next to the central base pair of the 13-base pair operator sequence. The surface of the KorB DNA-binding domain mirrors the overall acidity of KorB, whereas DNA binding occurs via a basic interaction surface. We present a model of KorB, including the structure of its dimerization domain, and discuss its interactions with the highly basic ParA homolog IncC.

Structural basis for selectivity in flavin-dependent monooxygenase-catalyzed oxidative dearomatization.

Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions and the use of environmentally benign reagents. These advantages have created a demand for biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the tradeoff that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. We recently demonstrated that a flavin-dependent monooxygenase, TropB, maintains high levels of site- and stereoselectivity across a range of structurally diverse substrates. Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of phenol substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by a crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data. In contrast to canonical class A FAD-dependent monooxygenases in which substrates bind in a protonated form, our data indicate that the phenolate form of the substrate binds in the active site. Furthermore, the substrate position is controlled through twopoint binding of the phenolate oxygen to Arg206 and Tyr239, which are shown to have distinct and essential roles in catalysis. Arg206 is involved in the reduction of the flavin cofactor, suggesting a role in flavin dynamics. Further, QM/MM simulations reveal the interactions that govern the facial selectivity that leads to a highly enantioselective transformation. Thus, the structural origins of the high levels of site-and stereoselectivity observed in reactions of TropB across a range of substrates are elucidated, providing a foundation for future protein engineering and reaction development efforts.

A novel reporter for real-time, quantitative imaging of AKT-directed K63-poly-ubiquitination in living cells.

Post-translational K63-linked poly-ubiquitination of AKT is required for its membrane recruitment and phosphorylation dependent activation in response to growth-factor stimulation. Current assays for target specific poly-ubiquitination involve cumbersome enzymatic preparations and semi-quantitative readouts. We have engineered a reporter that can quantitatively and in a target specific manner report on AKT-directed K63-polyubiquitination (K63UbR) in live cells. The reporter constitutes the AKT-derived poly-ubiquitination substrate peptide, a K63 poly-ubiquitin binding domain (UBD) as well as the split luciferase protein complementation domains. In cells, wherein signaling events upstream of AKT are activated (e.g. either EGFR or IGFR), poly-ubiquitination of the reporter leads to a stearic constraint that prevents luciferase complementation. However, upon inhibition of growth factor receptor signaling, loss of AKT poly-ubiquitination results in a decrease in interaction between the target peptide and the UBD, allowing for reconstitution of the split luciferase domains and therefore increased bioluminescence in a quantitative and dynamic manner. The K63UbR was confirmed to be suitable for high throughput screen (HTS), thus providing an excellent tool for small molecule or siRNA based HTS to discover new inhibitors or identify novel regulators of this key signaling node. Furthermore, the K63UbR platform could be adapted for non-invasive monitoring of additional target specific K63-polyubiquitination events in live cells.

The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition.

Sleeping Beauty (SB) is the most active Tc1/ mariner-type transposon in vertebrates. SB contains two transposase-binding sites (DRs) at the end of each terminal inverted repeat (IR), a feature termed the IR/DR structure. We investigated the involvement of cellular proteins in the regulation of SB transposition. Here, we establish that the DNA-bending, high-mobility group protein, HMGB1 is a host-encoded cofactor of SB transposition. Transposition was severely reduced in mouse cells deficient in HMGB1. This effect was rescued by transient over-expression of HMGB1, and was partially complemented by HMGB2, but not with the HMGA1 protein. Over-expression of HMGB1 in wild-type mouse cells enhanced transposition, indicating that HMGB1 can be a limiting factor of transposition. SB transposase was found to interact with HMGB1 in vivo, suggesting that the transposase may recruit HMGB1 to transposon DNA. HMGB1 stimulated preferential binding of the transposase to the DR further from the cleavage site, and promoted bending of DNA fragments containing the transposon IR. We propose that the role of HMGB1 is to ensure that transposase-transposon complexes are first formed at the internal DRs, and subsequently to promote juxtaposition of functional sites in transposon DNA, thereby assisting the formation of synaptic complexes.

Structural Basis for Cyclopropanation by a Unique Enoyl-Acyl Carrier Protein Reductase.

The natural product curacin A, a potent anticancer agent, contains a rare cyclopropane group. The five enzymes for cyclopropane biosynthesis are highly similar to enzymes that generate a vinyl chloride moiety in the jamaicamide natural product. The structural biology of this remarkable catalytic adaptability is probed with high-resolution crystal structures of the curacin cyclopropanase (CurF ER), an in vitro enoyl reductase (JamJ ER), and a canonical curacin enoyl reductase (CurK ER). The JamJ and CurK ERs catalyze NADPH-dependent double bond reductions typical of enoyl reductases (ERs) of the medium-chain dehydrogenase reductase (MDR) superfamily. Cyclopropane formation by CurF ER is specified by a short loop which, when transplanted to JamJ ER, confers cyclopropanase activity on the chimeric enzyme. Detection of an adduct of NADPH with the model substrate crotonyl-CoA provides indirect support for a recent proposal of a C2-ene intermediate on the reaction pathway of MDR enoyl-thioester reductases.

Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition.

Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.

RP4 repressor protein KorB binds to the major groove of the operator DNA: a Raman study.

KorB is a member of the ParB family of bacterial partitioning proteins. The protein encoded by the conjugative plasmid RP4 is part of the global control circuit and regulates the expression of plasmid genes, the products of which are involved in replication, transfer, and stable inheritance. KorB is a homodimeric protein which binds to palindromic 13 bp DNA sequences [5'-TTTAGC((G)/(C))GCTAAA-3'] present 12 times in the 60 kb plasmid. Each KorB subunit is composed of two domains; the C-domain is responsible for the dimerization of the protein, whereas the N-terminal domain recognizes and binds to the operator sequence (O(B)). Here we describe results of a Raman spectroscopic study of the interaction of the N-domain with a double-stranded model oligonucleotide composed of the palindromic binding sequence and terminal 5'-A(Br)U and AG-3' bases. Comparison of the Raman spectra of the free KorB N-domain and O(B) DNA with the spectrum of the complex reveals large differences. KorB-N binds in the major groove of the O(B) DNA, and the interactions induce changes in the DNA backbone and in the secondary structure of the protein.