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Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa., (© 2021. The Author(s).)

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.

The X-ray crystal structure of a salicylate hydroxylase from Pseudomonas putida S-1 complexed with coenzyme FAD has been determined to a resolution of 2.5 Å. Structural conservation with p- or m-hydroxybenzoate hydroxylase is very good throughout the topology, despite a low amino sequence identity of 20-40% between these three hydroxylases. Salicylate hydroxylase is composed of three distinct domains and includes FAD between domains I and II, which is accessible to solvent. In this study, which analyzes the tertiary structure of the enzyme, the unique reaction of salicylate, i.e. decarboxylative hydroxylation, and the structural roles of amino acids surrounding the substrate, are considered., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The phenylacetate degradation pathway is present in a wide range of microbes. A key component of this pathway is the four-subunit phenylacetyl-coenzyme A monooxygenase complex (PA-CoA MO, PaaACBE) that catalyzes the insertion of an oxygen in the aromatic ring of PA. This multicomponent enzyme represents a new family of monooxygenases. We have previously determined the structure of the PaaAC subcomplex of catalytic (A) and structural (C) subunits and shown that PaaACB form a stable complex. The PaaB subunit is unrelated to the small subunits of homologous monooxygenases and its role and organization of the PaaACB complex is unknown. From low-resolution crystal structure, electron microscopy and small angle X-ray scattering we show that the PaaACB complex forms heterohexamers, with a homodimer of PaaB bridging two PaaAC heterodimers. Modeling the interactions of reductase subunit PaaE with PaaACB suggested that a unique and conserved 'lysine bridge' constellation near the Fe-binding site in the PaaA subunit (Lys68, Glu49, Glu72 and Asp126) may form part of the electron transfer path from PaaE to the iron center. The crystal structure of the PaaA(K68Q/E49Q)-PaaC is very similar to the wild-type enzyme structure, but when combined with the PaaE subunit the mutant showed 20-50 times reduced activity, supporting the functional importance of the 'lysine bridge'., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine alpha-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation showed efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr(946), Ser(949)) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100-kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins.

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.

L-Lactate oxidase (LOX) from Aerococcus viridans catalyzes the oxidation of L-lactate to pyruvate by the molecular oxygen and belongs to a large family of 2-hydroxy acid-dependent flavoenzymes. To investigate the interaction of LOX with pyruvate in structural details and understand the chemical mechanism of flavin-dependent L-lactate dehydrogenation, the LOX-pyruvate complex was crystallized and the crystal structure of the complex has been solved at a resolution of 1.90 Angstrom. One pyruvate molecule bound to the active site and located near N5 position of FMN for subunits, A, B, and D in the asymmetric unit, were identified. The pyruvate molecule is stabilized by the interaction of its carboxylate group with the side-chain atoms of Tyr40, Arg181, His265, and Arg268, and of its keto-oxygen atom with the side-chain atoms of Tyr146, Tyr215, and His265. The alpha-carbon of pyruvate is found to be 3.13 Angstrom from the N5 atom of FMN at an angle of 105.4 degrees from the flavin N5-N10 axis.

Peptidylglycine-alpha-amidating monooxygenase (PAM) is a bi-functional enzyme known to catalyze the post-translational bioactivation of signaling peptides. Although PAM is highly concentrated within the cardiac atrium, this tissue does not produce appreciable amounts of alpha-amidated peptides and thus, the function of PAM in atrium remains largely unknown. In this study, we demonstrate that PAM co-localizes in atrial secretory granules with the storage form of atrial natriuretic peptide (pro-ANP, amino acids 1-126), a hormone involved in the maintenance of blood pressure and fluid homeostasis. ANP is not amidated by PAM, but rather is processed to its active form (amino acids 99-126) by the proteolytic cleavage of pro-ANP. We demonstrate here by subcellular fractionation and biochemical analyses that PAM co-localizes with pro-ANP in secretory granules, where together they constitute the two most abundant membrane-associated proteins, accounting for approximately 95% of the total granular membrane protein. Respectively, light and electron microscopic immunohistochemistry show intense staining for PAM in atrial cardiomyocyctes and subcellular localization of PAM to secretory granules. Additionally, we demonstrate that while pro-ANP is readily found in the soluble contents of the granule lumen, significant amounts remain tightly associated with the membranes even after vigorous washing and estimate the molar ratio of pro-ANP to PAM to be approximately 30:1 in the membrane fraction. We postulate here that the primary function of PAM in the atrium is structural rather than enzymatic. In this regard, PAM may contribute to the packaging of pro-ANP within the secretory granule and possibly function in the presentation of pro-ANP for proteolytic processing.

Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron microscopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45 degrees C. The addition of glycerol and dextran 500,000 to the enzyme sample enhanced storage stability.

MCD spectra of camphor-free and camphor-bound reduced cytochrome P450cam have been recorded for the near UV and visible spectral regions at temperatures from 300K down to 2.1K and compared with those of the carbon monoxide photoproducts generated at 4.2K. In the absence of camphor, the reduced P450 is spectroscopically different from the photoproduct. In the presence of camphor, however, the spectra of the reduced P450 and of the photoproduct are almost similar and behave like the photoproduct of the camphor-free enzyme. This behavior indicates that substrate binding induces a higher active site rigidity. From the significant alteration of the temperature dependence of the MCD intensity for the reduced enzyme induced by camphor binding it is concluded that the near degeneracy of the electronic ground state in the substrate-free enzyme is removed by substrate binding.

The alpha-ketoglutarate-dependent dioxygenase, L-asp(L-Asn)-beta-hydroxylase which posttranslationally hydroxylates specific aspartic acid (asparagine) residues within epidermal growth factor-like domains was purified from bovine liver and characterized. A 52-kDa and a 56-kDa species of this enzyme, which accounted for 60 and 30% of the total enzymatic activity, respectively, were purified to apparent homogeneity. Amino-terminal sequence analyses and immunoblots utilizing antisera raised to the intact 52-kDa species as well as to two complementary fragments of this species demonstrated that the 52- and 56-kDa species differ by a 22-amino acid amino-terminal extension. The remaining 10% of the purified enzymatic activity could be accounted for by the presence of immunologically related higher molecular mass forms (56-90 kDa) of L-Asp(L-Asn)-beta-hydroxylase. Strong evidence was obtained from the results of immunoextraction studies that L-Asp(L-Asn)-beta-hydroxylase can be identified with the purified proteins. Kinetic and physical studies suggest that L-Asp(L-Asn)-beta-hydroxylase exists as a monomer with a compact catalytic domain and an extended protease-sensitive amino terminus whose function remains to be determined. Since the purified L-Asp(L-Asn)-beta-hydroxylase hydroxylated both L-Asp- and L-Asn-containing substrates, it is possible that a single enzyme is responsible for the hydroxylation of Asp and Asn residues in vivo.