Fanglu Huang

Crystal structure and mechanism of a bacterial fluorinating enzyme

Fluorine is the thirteenth most abundant element in the earths crust, but fluoride concentrations in surface water are low and fluorinated metabolites are extremely rare. The fluoride ion is a potent nucleophile in its desolvated state, but is tightly hydrated in water and effectively inert. Low availability and a lack of chemical reactivity have largely excluded fluoride from biochemistry: in particular, fluorines high redox potential precludes the haloperoxidase-type mechanism used in the metabolic incorporation of chloride and bromide ions. But fluorinated chemicals are growing in industrial importance, with applications in pharmaceuticals, agrochemicals and materials products. Reactive fluorination reagents requiring specialist process technologies are needed in industry and, although biological catalysts for these processes are highly sought after, only one enzyme that can convert fluoride to organic fluorine has been described. Streptomyces cattleya can form carbon–fluorine bonds and must therefore have evolved an enzyme able to overcome the chemical challenges of using aqueous fluoride. Here we report the sequence and three-dimensional structure of the first native fluorination enzyme, 5′-fluoro-5′-deoxyadenosine synthase, from this organism. Both substrate and products have been observed bound to the enzyme, enabling us to propose a nucleophilic substitution mechanism for this biological fluorination reaction.

Spectroscopic and electronic structure studies of aromatic electrophilic attack and hydrogen-atom abstraction by non-heme iron enzymes

(4-Hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are two α-keto acid dependent mononuclear non-heme iron enzymes that use the same substrate, (4-hydroxyphenyl)pyruvate, but exhibit two different general reactivities. HmaS performs hydrogen-atom abstraction to yield benzylic hydroxylated product (S)-(4-hydroxy)mandelate, whereas HPPD utilizes an electrophilic attack mechanism that results in aromatic hydroxylated product homogentisate. These enzymes provide a unique opportunity to directly evaluate the similarities and differences in the reaction pathways used for these two reactivities. An FeII methodology using CD, magnetic CD, and variable-temperature, variable-field magnetic CD spectroscopies was applied to HmaS and compared with that for HPPD to evaluate the factors that affect substrate interactions at the active site and to correlate these to the different reactivities exhibited by HmaS and HPPD to the same substrate. Combined with density functional theory calculations, we found that HmaS and HPPD have similar substrate-bound complexes and that the role of the protein pocket in determining the different reactivities exhibited by these enzymes (hydrogen-atom abstraction vs. aromatic electrophilic attack) is to properly orient the substrate, allowing for ligand field geometric changes along the reaction coordinate. Elongation of the FeIVO bond in the transition state leads to dominant FeIIIO•− character, which significantly contributes to the reactivity with either the aromatic π-system or the CH σ-bond.

Elaboration of Neosamine Rings in the Biosynthesis of Neomycin and Butirosin

The proteins Neo‐11 and Neo‐18 encoded in the neomycin gene cluster (neo) of Streptomyces fradiae NCIMB 8233 have been characterized as glucosaminyl‐6′‐oxidase and 6′‐oxoglucosaminyl:L‐glutamate aminotransferase, respectively. The joint activity of Neo‐11 and Neo‐18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD‐dependent dehydrogenation followed by a pyridoxal‐5′‐phosphate‐mediated transamination. Neo‐18 is also shown to catalyze deamination at C‐6′′′ of neomycin, thus suggesting bifunctional roles of the two enzymes in the formation of both neosamine rings of neomycin. The product of the btrB gene, a homologue of neo‐18 in the butirosin biosynthetic gene cluster (btr) in Bacillus circulans, exhibits the same activity as Neo‐18; this indicates that there is a similar reaction sequence in both butirosin and neomycin biosynthesis.

Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution

Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non‐heme iron‐dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.

Delineating the Biosynthesis of Gentamicin X2, the Common Precursor of the Gentamicin C Antibiotic Complex

Summary Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.

Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr42, His76, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg120 located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

Specificity and promiscuity at the branch point in gentamicin biosynthesis.

Summary Gentamicin C complex is a mixture of aminoglycoside antibiotics used to treat severe Gram-negative bacterial infections. We report here key features of the late-stage biosynthesis of gentamicins. We show that the intermediate gentamicin X2, a known substrate for C-methylation at C-6′ to form G418 catalyzed by the radical SAM-dependent enzyme GenK, may instead undergo oxidation at C-6′ to form an aldehyde, catalyzed by the flavin-linked dehydrogenase GenQ. Surprisingly, GenQ acts in both branches of the pathway, likewise oxidizing G418 to an analogous ketone. Amination of these intermediates, catalyzed mainly by aminotransferase GenB1, produces the known intermediates JI-20A and JI-20B, respectively. Other pyridoxal phosphate-dependent enzymes (GenB3 and GenB4) act in enigmatic dehydroxylation steps that convert JI-20A and JI-20B into the gentamicin C complex or (GenB2) catalyze the epimerization of gentamicin C2a into gentamicin C2.

Crystal Structures of the Plp- and Pmp-Bound Forms of Btrr, a Dual Functional Aminotransferase Involved in Butirosin Biosynthesis

The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2‐deoxystreptamine (2‐DOS)‐containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)‐dependent transamination reaction both of 2‐deoxy‐scyllo‐inosose to 2‐deoxy‐scyllo‐inosamine and of amino‐dideoxy‐scyllo‐inosose to 2‐DOS. The high‐resolution crystal structures of the PLP‐ and PMP‐bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 Å and 1.7 Å with Rfactor/Rfree values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an α helical N‐terminus, a central αβα sandwich domain and an αβ C‐terminal domain. The structures of the PLP‐ and PMP‐bound enzymes are very similar; however BtrR‐PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side‐chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three‐dimensional structure of an enzyme from the butirosin biosynthesis gene cluster. Proteins 2006.

Biosynthesis of aminoglycoside antibiotics: cloning, expression and characterisation of an aminotransferase involved in the pathway to 2-deoxystreptamine.

The gene btrR from Bacillus circulans has been cloned and expressed and shown to produce a protein which catalyses the transamination of 2-deoxy-scyllo-inosose to give 2-deoxy-scyllo-inosamine, an intermediate in the biosynthesis of 2-deoxystreptamine.

Insights into 6‐Methylsalicylic Acid Bio‐assembly by Using Chemical Probes

Abstract Chemical probes capable of reacting with KS (ketosynthase)‐bound biosynthetic intermediates were utilized for the investigation of the model type I iterative polyketide synthase 6‐methylsalicylic acid synthase (6‐MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6‐MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6‐MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities.