Active-Site Structure of a β-Hydroxylase in Antibiotic Biosynthesis

Vu, Van V.; Makris, Thomas M.; Lipscomb, John D.; Que, Lawrence

doi:10.1021/ja201822v

Cited by 22 publications

(36 citation statements)

References 45 publications

(86 reference statements)

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“…The Fourier transform exhibits a strong inner shell feature at r′ ∼1.5 Å, a second shell feature at r′ ∼2.3 Å, and third shell features between r′ = 2.3 and 3.7 Å. The third shell features in the Fourier transform correspond to the double-humped feature centered at ∼4 Å −1 in the EXAFS spectrum, which arises from the multiple scattering paths of several imidazole moieties as found in the spectra of many other metalloproteins and model complexes (33)(34)(35)(36)(37). Fitting progress is described in SI Appendix, Table S5.…”

Section: Resultsmentioning

confidence: 84%

A family of starch-active polysaccharide monooxygenases

Beeson

Span

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.copper enzymes | oxygen activation | CBM20 P olysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species (1-5). They have recently been found to oxidatively degrade chitin (6-8) and cellulose (8)(9)(10)(11)(12)(13)(14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10)(11)(12)14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Several fungal PMOs were shown to significantly enhance the degradation of cellulose by hydrolytic cellulases (9), indicating that these enzymes can be used in the conversion of plant biomass into biofuels and other renewable chemicals.There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9-12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6, 8) or cellulose (8, 13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel β-sandwich core and a highly conserved ...

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Section: Resultsmentioning

confidence: 84%

A family of starch-active polysaccharide monooxygenases

Beeson

Span

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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236

215

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“…The hydroxide bridge-Fe bond distances are 1.9 Å and 2.1 Å for Fe1 and Fe2, respectively, whereas both μ-oxo bridge-Fe bond distances in the as-isolated structure are roughly 1.8 Å. 5, 19 The overall increase in iron-ligand bond distances for the chemically reduced cluster is expected upon reduction of both iron ions to the ferrous state. The cluster retains the μ-1,1-bridging D403 carboxylate.…”

Section: Resultsmentioning

confidence: 99%

“…We have shown previously that X-ray absorption spectroscopy can provide structural insight into the diiron cluster of CmlA. 19 Here we use this technique to study the nature of the complex between reduced CmlA and a functional CmlP variant (CmlP AT ) with and without L-PAPA covalently attached. This approach reveals a specific diiron cluster carboxylate ligand that appears to regulate reactivity with O 2 .…”

Section: Introductionmentioning

confidence: 99%

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase

Jasniewski

Knoot

Lipscomb³

et al. 2016

Biochemistry

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The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor L-para-aminophenylalanine (L-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster which is reduced to the diferrous state (WT R ) to initiate O 2 activation. However, rapid O 2 activation only occurs when WT R is bound to CmlP, the NRPS to which L-PAPA is covalently attached. Here the X-ray crystal structure of WT R is reported, which is very similar to that of the as-isolated diferric enzyme in which the irons are coordinately saturated. X-ray absorption spectroscopy is used to investigate the WT R cluster ligand structure as well as the structures of WT R in complex with a functional CmlP variant (CmlP AT ) with and without L-PAPA attached. It is found that formation of the active WT R -CmlP AT~L -PAPA complex converts at least one iron of the cluster from six-to five-coordinate by changing a bidentately bound amino acid carboxylate to monodentate on Fe1. The only bidentate carboxylate in the structure of WT R is E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O 2 in the presence or absence of CmlP AT~L -PAPA, showing loss of regulation. However this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O 2 reactivity with juxtaposition of the substrate and the reactive oxygen species. The carboxylate shift in response to substrate binding represents a novel regulatory strategy for oxygen activation in diiron oxygenases. Graphical abstract * Corresponding Authors

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“…For all five of the hDOHH samples in this study, there are scatterers included in the fits at distances ranging from 3.1 – 4.3 Å that are likely derived from the imidazole rings of the histidine ligands, as observed for the diiron sites in sMMO and ToMO [37, 98], methemerythrin and RNR R2 [99], and CmlA [100]. Histidine ligands typically give rise to scatterers at 3.1 and 4.3 Å, which are respectively associated with the C atoms adjacent to the coordinated N atom and the C and N atoms further away (Figure 3).…”

Section: Discussionmentioning

confidence: 99%

X-ray absorption spectroscopic characterization of the diferric-peroxo intermediate of human deoxyhypusine hydroxylase in the presence of its substrate eIF5a

Jasniewski

Engstrom

et al. 2016

J Biol Inorg Chem

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Human deoxyhypusine hydroxylase (hDOHH) is an enzyme that is involved in the critical post-translational modification of the eukaryotic translation initiation factor 5A (eIF5A). Following the conversion of a lysine residue on eIF5A to deoxyhypusine (Dhp) by deoxyhypusine synthase, hDOHH hydroxylates Dhp to yield the unusual amino acid residue hypusine (Hpu), a modification that is essential for eIF5A to promote peptide synthesis at the ribosome, among other functions. Purification of hDOHH overexpressed in E. coli affords enzyme that is blue in color, a feature that has been associated with the presence of a peroxo-bridged diiron(III) active site. To gain further insight into the nature of the diiron site and how it may change as hDOHH goes through the catalytic cycle, we have conducted X-ray absorption spectroscopic studies of hDOHH on five samples that represent different species along its reaction pathway. Structural analysis of each species has been carried out, starting with the reduced diferrous state, proceeding through its O2 adduct, and ending with a diferric decay product. Our results show that the Fe•••Fe distances found for the five samples fall within a narrow range of 3.4–3.5 Å, suggesting that hDOHH has a fairly constrained active site. This pattern differs significantly from what has been associated with canonical dioxygen activating nonheme diiron enzymes such as soluble methane monooxygenase and Class 1A ribonucleotide reductases, for which the Fe•••Fe distance can change by as much as 1 Å during the redox cycle. These results suggest that the O2 activation mechanism for hDOHH deviates somewhat from that associated with the canonical nonheme diiron enzymes, opening the door to new mechanistic possibilities for this intriguing family of enzymes.

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Active-Site Structure of a β-Hydroxylase in Antibiotic Biosynthesis

Cited by 22 publications

References 45 publications

A family of starch-active polysaccharide monooxygenases

A family of starch-active polysaccharide monooxygenases

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase

X-ray absorption spectroscopic characterization of the diferric-peroxo intermediate of human deoxyhypusine hydroxylase in the presence of its substrate eIF5a

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