A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

Characterization of a phylogenetically distinct extradiol dioxygenase involved in the bacterial catabolism of lignin-derived aromatic compounds.

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM ∼106 M-1 s-1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates' binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate's alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.

A scoping review of resting-state brain functional alterations in Type 2 diabetes.

Resting-state functional magnetic resonance imaging (rs-fMRI) has been actively used in the last decade to investigate brain functional connectivity alterations in Type 2 Diabetes Mellitus (T2DM) to understand the neuropathophysiology of T2DM in cognitive degeneration. Given the emergence of new analysis techniques, this scoping review aims to map the rs-fMRI analysis techniques that have been applied in the literature and reports the latest rs-fMRI findings that have not been covered in previous reviews. Graph theory, the contemporary rs-fMRI analysis, has been used to demonstrate altered brain topological organisations in people with T2DM, which included altered degree centrality, functional connectivity strength, the small-world architecture and network-based statistics. These alterations were correlated with T2DM patients' cognitive performances. Graph theory also contributes to identify unbiased seeds for seed-based analysis. The expanding rs-fMRI analytical approaches continue to provide new evidence that helps to understand the mechanisms of T2DM-related cognitive degeneration.

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly.

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.

Sequence Divergence in the Arginase Domain of Ornithine Decarboxylase/Arginase in Fusobacteriacea Leads to Loss of Function in Oral Associated Species.

A number of species within the Fusobacteriaceae family of Gram-negative bacteria uniquely encode for an ornithine decarboxylase/arginase (ODA) that ostensibly channels l-ornithine generated by hydrolysis of l-arginine to putrescine formation. However, two aspartate residues required for coordination to a catalytically obligatory manganese cluster of arginases are substituted for a serine and an asparagine. Curiously, these natural substitutions occur only in a clade of Fusobacterium species that inhabit the oral cavity. Herein, we expressed and isolated full-length ODA from the opportunistic oral pathogen Fusobacterium nucleatum along with the individual arginase and ornithine decarboxylase components. The crystal structure of the arginase domain reveals that it adopts the classical α/ß arginase-fold, but metal ions are absent in the active site. As expected, the ureohydrolase activity with l-arginine was not detected for wild-type ODA or the isolated arginase domain. However, engineering of the complete metal coordination environment through site-directed mutagenesis restored Mn2+ binding capacity and arginase activity, although the catalytic efficiency for l-arginine was low (60-100 M-1 s-1). Full-length ODA and the isolated ODC component were able to decarboxylate both l-ornithine and l-arginine to form putrescine and agmatine, respectively, but kcat/KM of l-ornithine was ∼20-fold higher compared to l-arginine. We discuss environmental conditions that may have led to the natural selection of an inactive arginase in the oral associated species of Fusobacterium.

Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6.

Lignostilbene-α,ß-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from ß-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M-1 s-1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Soft-Dielectron Excess in Proton-Proton Collisions at sqrt[s]=13 TeV.

A measurement of dielectron production in proton-proton (pp) collisions at sqrt[s]=13 TeV, recorded with the ALICE detector at the CERN LHC, is presented in this Letter. The data set was recorded with a reduced magnetic solenoid field. This enables the investigation of a kinematic domain at low dielectron (ee) invariant mass m_{ee} and pair transverse momentum p_{T,ee} that was previously inaccessible at the LHC. The cross section for dielectron production is studied as a function of m_{ee}, p_{T,ee}, and event multiplicity dN_{ch}/dη. The expected dielectron rate from hadron decays, called hadronic cocktail, utilizes a parametrization of the measured η/π^{0} ratio in pp and proton-nucleus collisions, assuming that this ratio shows no strong dependence on collision energy at low transverse momentum. Comparison of the measured dielectron yield to the hadronic cocktail at 0.15

Elliptic Flow of Electrons from Beauty-Hadron Decays in Pb-Pb Collisions at sqrt[s_{NN}]=5.02 TeV.

The elliptic flow of electrons from beauty hadron decays at midrapidity (|y|<0.8) is measured in Pb-Pb collisions at sqrt[s_{NN}]=5.02 TeV with the ALICE detector at the LHC. The azimuthal distribution of the particles produced in the collisions can be parametrized with a Fourier expansion, in which the second harmonic coefficient represents the elliptic flow, v_{2}. The v_{2} coefficient of electrons from beauty hadron decays is measured for the first time in the transverse momentum (p_{T}) range 1.3-6 GeV/c in the centrality class 30%-50%. The measurement of electrons from beauty-hadron decays exploits their larger mean proper decay length cτ≈500 µm compared to that of charm hadrons and most of the other background sources. The v_{2} of electrons from beauty hadron decays at midrapidity is found to be positive with a significance of 3.75 σ. The results provide insights into the degree of thermalization of beauty quarks in the medium. A model assuming full thermalization of beauty quarks is strongly disfavored by the measurement at high p_{T}, but is in agreement with the results at low p_{T}. Transport models including substantial interactions of beauty quarks with an expanding strongly interacting medium describe the measurement within uncertainties.

A qualitative exploration of the experiences of school nurses during COVID-19 pandemic as the frontline primary health care professionals.

BACKGROUND: It is widely acknowledged that the experiences of frontline primary health care professionals during COVID-19 are important to understand how they respond and act under situations of pandemic as the gatekeepers in primary health care system. School nurses are primary health care professionals who lead health care in schools and practice in a holistic manner to address the needs of schoolchildren and school personnel. There are rising mental health concerns of frontline health care professionals with anxiety and panic disorders, somatic symptoms, and feeling isolated. No studies use a qualitative study approach to document community frontline school nursing professionals' experiences and challenges during the COVID-19 pandemic. Hence, understanding the school nurses' experiences and challenges to fight against COVID-19 in the communities is important. PURPOSE: This study aims to explore the experiences of school nurses during the COVID-19 pandemic in Hong Kong. METHODS: A qualitative study design adopted the principles of thematic analysis. Nineteen school nurses were recruited to participate in individual semistructured interviews and shared their roles and responsibilities during the COVID-19 pandemic. FINDINGS: Three themes indicated the school nurses' expand professional responsibilities to fight against COVID-19 emerged from the data analysis. These were "Managing Stress," "Navigating the School Through the Pandemic," and "Raising the Profile of the School Nurse Professional," DISCUSSION: Findings reveal the important role of school nursing professionals in minimizing the community-wide risk posed by pandemics and the need to integrate them into planning and implementation of school health policies and guidelines in the primary health care system. This essential role in schools is necessary to assess, implement, monitor, prevent, and reduce the spread of virus in school communities and to minimize the burden to and extra health care resources utilized in the acute care setting during COVID-19 pandemic.

Probing the Effects of Strong Electromagnetic Fields with Charge-Dependent Directed Flow in Pb-Pb Collisions at the LHC.

The first measurement at the LHC of charge-dependent directed flow (v_{1}) relative to the spectator plane is presented for Pb-Pb collisions at sqrt[s_{NN}]=5.02 TeV. Results are reported for charged hadrons and D^{0} mesons for the transverse momentum intervals p_{T}>0.2 GeV/c and 3

Evidence of Spin-Orbital Angular Momentum Interactions in Relativistic Heavy-Ion Collisions.

The first evidence of spin alignment of vector mesons (K^{*0} and ϕ) in heavy-ion collisions at the Large Hadron Collider (LHC) is reported. The spin density matrix element ρ_{00} is measured at midrapidity (|y|<0.5) in Pb-Pb collisions at a center-of-mass energy (sqrt[s_{NN}]) of 2.76 TeV with the ALICE detector. ρ_{00} values are found to be less than 1/3 (1/3 implies no spin alignment) at low transverse momentum (p_{T}<2 GeV/c) for K^{*0} and ϕ at a level of 3σ and 2σ, respectively. No significant spin alignment is observed for the K_{S}^{0} meson (spin=0) in Pb-Pb collisions and for the vector mesons in pp collisions. The measured spin alignment is unexpectedly large but qualitatively consistent with the expectation from models which attribute it to a polarization of quarks in the presence of angular momentum in heavy-ion collisions and a subsequent hadronization by the process of recombination.

Measurement of the Low-Energy Antideuteron Inelastic Cross Section.

In this Letter, we report the first measurement of the inelastic cross section for antideuteron-nucleus interactions at low particle momenta, covering a range of 0.3≤p<4 GeV/c. The measurement is carried out using p-Pb collisions at a center-of-mass energy per nucleon-nucleon pair of sqrt[s_{NN}]=5.02 TeV, recorded with the ALICE detector at the CERN LHC and utilizing the detector material as an absorber for antideuterons and antiprotons. The extracted raw primary antiparticle-to-particle ratios are compared to the results from detailed ALICE simulations based on the geant4 toolkit for the propagation of (anti)particles through the detector material. The analysis of the raw primary (anti)proton spectra serves as a benchmark for this study, since their hadronic interaction cross sections are well constrained experimentally. The first measurement of the inelastic cross section for antideuteron-nucleus interactions averaged over the ALICE detector material with atomic mass numbers ⟨A⟩=17.4 and 31.8 is obtained. The measured inelastic cross section points to a possible excess with respect to the Glauber model parametrization used in geant4 in the lowest momentum interval of 0.3≤p<0.47 GeV/c up to a factor 2.1. This result is relevant for the understanding of antimatter propagation and the contributions to antinuclei production from cosmic ray interactions within the interstellar medium. In addition, the momentum range covered by this measurement is of particular importance to evaluate signal predictions for indirect dark-matter searches.

Scattering Studies with Low-Energy Kaon-Proton Femtoscopy in Proton-Proton Collisions at the LHC.

The study of the strength and behavior of the antikaon-nucleon (K[over ¯]N) interaction constitutes one of the key focuses of the strangeness sector in low-energy quantum chromodynamics (QCD). In this Letter a unique high-precision measurement of the strong interaction between kaons and protons, close and above the kinematic threshold, is presented. The femtoscopic measurements of the correlation function at low pair-frame relative momentum of (K^{+}p⊕K^{-}p[over ¯]) and (K^{-}p⊕K^{+}p[over ¯]) pairs measured in pp collisions at sqrt[s]=5, 7, and 13 TeV are reported. A structure observed around a relative momentum of 58 MeV/c in the measured correlation function of (K^{-}p⊕K^{+}p[over ¯]) with a significance of 4.4σ constitutes the first experimental evidence for the opening of the (K[over ¯]^{0}n⊕K^{0}n[over ¯]) isospin breaking channel due to the mass difference between charged and neutral kaons. The measured correlation functions have been compared to Jülich and Kyoto models in addition to the Coulomb potential. The high-precision data at low relative momenta presented in this work prove femtoscopy to be a powerful complementary tool to scattering experiments and provide new constraints above the K[over ¯]N threshold for low-energy QCD chiral models.

Structure-function analyses reveal key features in Staphylococcus aureus IsdB-associated unfolding of the heme-binding pocket of human hemoglobin.

IsdB is a receptor on the surface of the bacterial pathogen Staphylococcus aureus that extracts heme from hemoglobin (Hb) to enable growth on Hb as a sole iron source. IsdB is critically important both for in vitro growth on Hb and in infection models and is also highly up-regulated in blood, serum, and tissue infection models, indicating a key role of this receptor in bacterial virulence. However, structural information for IsdB is limited. We present here a crystal structure of a complex between human Hb and IsdB. In this complex, the α subunits of Hb are refolded with the heme displaced to the interface with IsdB. We also observe that atypical residues of Hb, His58 and His89 of αHb, coordinate to the heme iron, which is poised for transfer into the heme-binding pocket of IsdB. Moreover, the porphyrin ring interacts with IsdB residues Tyr440 and Tyr444 Previously, Tyr440 was observed to coordinate heme iron in an IsdB·heme complex structure. A Y440F/Y444F IsdB variant we produced was defective in heme transfer yet formed a stable complex with Hb (Kd = 6 ± 2 µm) in solution with spectroscopic features of the bis-His species observed in the crystal structure. Haptoglobin binds to a distinct site on Hb to inhibit heme transfer to IsdB and growth of S. aureus, and a ternary complex of IsdB·Hb·Hp was observed. We propose a model for IsdB heme transfer from Hb that involves unfolding of Hb and heme iron ligand exchange.

Investigations of Anisotropic Flow Using Multiparticle Azimuthal Correlations in pp, p-Pb, Xe-Xe, and Pb-Pb Collisions at the LHC.

Measurements of anisotropic flow coefficients (v_{n}) and their cross-correlations using two- and multiparticle cumulant methods are reported in collisions of pp at sqrt[s]=13 TeV, p-Pb at a center-of-mass energy per nucleon pair sqrt[s_{NN}]=5.02 TeV, Xe-Xe at sqrt[s_{NN}]=5.44 TeV, and Pb-Pb at sqrt[s_{NN}]=5.02 TeV recorded with the ALICE detector. The multiplicity dependence of v_{n} is studied in a very wide range from 20 to 3000 particles produced in the midrapidity region |η|<0.8 for the transverse momentum range 0.2v_{3}>v_{4} is found in pp and p-Pb collisions, similar to that seen in large collision systems, while a weak v_{2} multiplicity dependence is observed relative to nucleus-nucleus collisions in the same multiplicity range. Using a novel subevent method, v_{2} measured with four-particle cumulants is found to be compatible with that from six-particle cumulants in pp and p-Pb collisions. The magnitude of the correlation between v_{n}^{2} and v_{m}^{2}, evaluated with the symmetric cumulants SC(m,n) is observed to be positive at all multiplicities for v_{2} and v_{4}, while for v_{2} and v_{3} it is negative and changes sign for multiplicities below 100, which may indicate a different v_{n} fluctuation pattern in this multiplicity range. The observed long-range multiparticle azimuthal correlations in high multiplicity pp and p-Pb collisions can neither be described by pythia 8 nor by impact-parameter-Glasma, music, and ultrarelativistic quantum molecular dynamics model calculations, and hence, provide new insights into the understanding of collective effects in small collision systems.

First Observation of an Attractive Interaction between a Proton and a Cascade Baryon.

This Letter presents the first experimental observation of the attractive strong interaction between a proton and a multistrange baryon (hyperon) Ξ^{-}. The result is extracted from two-particle correlations of combined p-Ξ^{-}⊕p[over ¯]-Ξ[over ¯]^{+} pairs measured in p-Pb collisions at sqrt[s_{NN}]=5.02 TeV at the LHC with ALICE. The measured correlation function is compared with the prediction obtained assuming only an attractive Coulomb interaction and a standard deviation in the range [3.6, 5.3] is found. Since the measured p-Ξ^{-}⊕p[over ¯]-Ξ[over ¯]^{+} correlation is significantly enhanced with respect to the Coulomb prediction, the presence of an additional, strong, attractive interaction is evident. The data are compatible with recent lattice calculations by the HAL-QCD Collaboration, with a standard deviation in the range [1.8, 3.7]. The lattice potential predicts a shallow repulsive Ξ^{-} interaction within pure neutron matter and this implies stiffer equations of state for neutron-rich matter including hyperons. Implications of the strong interaction for the modeling of neutron stars are discussed.

Azimuthal Anisotropy of Heavy-Flavor Decay Electrons in p-Pb Collisions at sqrt[s_{NN}]=5.02 TeV.

Angular correlations between heavy-flavor decay electrons and charged particles at midrapidity (|η|<0.8) are measured in p-Pb collisions at sqrt[s_{NN}]=5.02 TeV. The analysis is carried out for the 0%-20% (high) and 60%-100% (low) multiplicity ranges. The jet contribution in the correlation distribution from high-multiplicity events is removed by subtracting the distribution from low-multiplicity events. An azimuthal modulation remains after removing the jet contribution, similar to previous observations in two-particle angular correlation measurements for light-flavor hadrons. A Fourier decomposition of the modulation results in a positive second-order coefficient (v_{2}) for heavy-flavor decay electrons in the transverse momentum interval 1.5

Measurement of ϒ(1S) Elliptic Flow at Forward Rapidity in Pb-Pb Collisions at sqrt[s_{NN}]=5.02 TeV.

The first measurement of the ϒ(1S) elliptic flow coefficient (v_{2}) is performed at forward rapidity (2.5

The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/ß-hydrolase superfamily.

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s-1 and 2.5 ± 0.2 × 107 m-1 s-1, respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/ß-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-ß-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C-C-hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.