Xiao-Xue Yan

AidH, an alpha/beta-hydrolase fold family member from an Ochrobactrum sp. strain, is a novel N-acylhomoserine lactonase.

ABSTRACT N-Acylhomoserine lactones (AHLs) are signaling molecules in many quorum-sensing (QS) systems that regulate interactions between various pathogenic bacteria and their hosts. Quorum quenching by the enzymatic inactivation of AHLs holds great promise in preventing and treating infections, and several such enzymes have been reported. In this study, we report the characterization of a novel AHL-degrading protein from the soil bacterium Ochrobactrum sp. strain T63. This protein, termed AidH, shares no similarity with any of the known AHL degradases but is highly homologous with a hydrolytic enzyme from Ochrobactrum anthropi ATCC 49188 that contains the alpha/beta-hydrolase fold. By liquid chromatography-mass spectrometry (MS) analysis, we demonstrate that AidH functions as an AHL-lactonase that hydrolyzes the ester bond of the homoserine lactone ring of AHLs. Mutational analyses indicate that the G-X-Nuc-X-G motif or the histidine residue conserved among alpha/beta-hydrolases is critical for the activity of AidH. Furthermore, the AHL-inactivating activity of AidH requires Mn2+ but not several other tested divalent cations. We also showed that AidH significantly reduces biofilm formation by Pseudomonas fluorescens 2P24 and the pathogenicity of Pectobacterium carotovorum, indicating that this enzyme is able to effectively quench QS-dependent functions in these bacteria by degrading AHLs.

From Structure to Function: Insights into the Catalytic Substrate Specificity and Thermostability Displayed by Bacillus subtilis Mannanase BCman

BCman, a beta-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-beta-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the beta4 and beta7 strands, respectively. The 1.45-A crystal structure reveals that BCman is a typical (beta/alpha)(8) folding type. One large difference from the saddle-shaped active center of other endo-beta-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+1 and -1) within the active site of BCman. These two sites are involved in the enzymes mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzymes stability.

High-resolution structures of AidH complexes provide insights into a novel catalytic mechanism for N-acyl homoserine lactonase

Crystal structures of the AHL-lactonase AidH in complex with substrate and product are reported at high resolution and a catalytic mechanism is proposed for the metal-independent AHL-lactonase.

Structure of HsdS subunit from Thermoanaerobacter tengcongensis sheds lights on mechanism of dynamic opening and closing of type I methyltransferase.

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase.

Crystal structure of human phosphomavelonate kinase at 1.8 A resolution

The mevalonate pathway of isoprenoid biosynthesis is the most chemically diverse pathway in nature. This pathway produces isopentenyl diphosphate,1 the fundamental fivecarbon building block for the biosynthesis of over 23,000 isoprenoid compounds,2 such as sterols,3 caroteniods,4 dolichols,5 ubiquinone,6 and some prominent classes of prenylated proteins.7 These compounds are essential for cell growth and differentiation, gene expression, protein glycosylation, and cytoskeletal assembly. In addition, isoprenoid compounds are essential for posttranslational modification of proteins involved in intracellular signaling.8,9 Phosphomevalonate kinase (PMK, EC2.7.4.2) catalyzes the rate-limiting step for biosynthesis of isopentenyl diphosphate from mevalonate. Specifically, PMK catalyzes the transfer of the g-phosphoryl group of ATP to (R)-5phosphomevalonate (Pmev), which results in the formation of ADP and (R)-5-diphosphomevalonate (DPM). There are two nonorthologous types of PMK. One orthologue of the Saccharomyces cerevisiae ERG8 gene is present in eubacteria, fungi, and plants, whereas orthologues of human PMK (hPMK) gene are found only in animals and low-homology invertebrates.10 Examination of the PMK crystal structure in Streptococcus pneumoniae confirmed that it belongs to the GHMP kinase (galactokinase/homoserine kinase/mevalonate kinase/phosphomevalonate) superfamily.11 However, no empirical threedimensional structural information is available for the divergent animal PMK proteins. Animal PMKs have been purified from a variety of tissues, and several animal PMK genes have been identified. The hPMK gene was first cloned in 1996 when Chambliss et al. screened the human liver library with the porcine PMK clone.12 They first expressed a GSThuman PMK fusion construct in bacteria and did some preliminary characterization. Recently, Herdendorf et al. continued functional studies with the recombinant His-tag hPMK, alongside several mutant forms. Results from these kinetic and biophysical studies suggested that the hPMK belongs to the nucleoside monophosphate kinase (NMP) family, and possibly contains a ‘‘Walker A’’ motif.13,14 The precise structure of hPMK is necessary to facilitate a more detailed understanding of diphosphomevalonate synthesis in humans. Here we report high-resolution crystal structure of hPMK at 1.76 Å. The three-dimensional structure of hPMK reveals that the enzyme is classified as a member of the NMP superfamily. Ours is the first report of hPMK crystal structure that suggests a potential substrate binding site and a possible enzyme catalytic mechanism.

Structural basis underlying complex assembly and conformational transition of the type I R-M system

Significance Type I restriction-modification (R-M) enzymes are large molecular machines found in the majority of bacterial species. They can add methylation modifications to the self-DNA and degrade the invading unmodified DNA. The lack of high-resolution structures of type I R-M complexes impairs our understanding of the mechanism of subunit assembly and conformational transition. Here we report the first high-resolution structure of the type I MTase complex in its “open” conformation, including one DNA-recognition subunit, two DNA-modification subunits, one bound DNA, and two S-adenosyl methionine cofactors. We propose an updated model for the complex assembly and conformational transition. The structural and biochemical characterization of the type I R-M system reported in this study provides guidelines for future applications in molecular biology. Type I restriction-modification (R-M) systems are multisubunit enzymes with separate DNA-recognition (S), methylation (M), and restriction (R) subunits. Despite extensive studies spanning five decades, the detailed molecular mechanisms underlying subunit assembly and conformational transition are still unclear due to the lack of high-resolution structural information. Here, we report the atomic structure of a type I MTase complex (2M+1S) bound to DNA and cofactor S-adenosyl methionine in the “open” form. The intermolecular interactions between M and S subunits are mediated by a four-helix bundle motif, which also determines the specificity of the interaction. Structural comparison between open and previously reported low-resolution “closed” structures identifies the huge conformational changes within the MTase complex. Furthermore, biochemical results show that R subunits prefer to load onto the closed form MTase. Based on our results, we proposed an updated model for the complex assembly. The work reported here provides guidelines for future applications in molecular biology.

RecOR complex including RecR N-N dimer and RecO monomer displays a high affinity for ssDNA

RecR is an important recombination mediator protein in the RecFOR pathway. RecR together with RecO and RecF facilitates RecA nucleoprotein filament formation and homologous pairing. Structural and biochemical studies of Thermoanaerobacter tengcongensis RecR (TTERecR) and its series mutants revealed that TTERecR uses the N-N dimer as a basic functional unit to interact with TTERecO monomer. Two TTERecR N-N dimers form a ring-shaped tetramer via an interaction between their C-terminal regions. The tetramer is a result of crystallization only. Hydrophobic interactions between the entire helix-hairpin-helix domains within the N-terminal regions of two TTERecR monomers are necessary for formation of a RecR functional N-N dimer. The TTERecR N-N dimer conformation also affects formation of a hydrophobic patch, which creates a binding site for TTERecO in the TTERecR Toprim domain. In addition, we demonstrate that TTERecR does not bind single-stranded DNA (ssDNA) and binds double-stranded DNA very weakly, whereas TTERecOR complex can stably bind DNA, with a higher affinity for ssDNA than double-stranded DNA. Based on these results, we propose an interaction model for the RecOR:ssDNA complex.

Crystal structure of a putative methylmalonyl-coenzyme a epimerase from Thermoanaerobacter tengcongensis at 2.0 A resolution

Methylmalonyl-CoA is an important metabolic intermediate that exists in several key degradative and biosynthetic pathways.1 The pathway responsible for the degradation of branched amino acids and odd chain fatty acids involves three steps. In the first step, a biotindependent carboxylation produces the S-epimer of methylmalonyl-CoA. The third step, the B12-dependent methylmalonyl-CoA mutase converts the R-epimer of methylmalonyl-CoA to succinyl-CoA.1 This key epimerization between the two steps is carried out by the essential enzyme methylmalonyl-CoA epimerase (MMCE: EC number 5.1.99.1), which catalyzes the conversion of (2S)methylmalonyl-CoA to (2R)-methylmalonyl-CoA. In prokaryotes, MMCE participated in autotrophic CO2 fixation via the 3-hydroxypropionate pathway, in propionate fermentation, the regeneration of glyoxylate and in the biosynthesis of polyketide antibiotics.2–6 In animals, defects of this pathway can result in a buildup of methylmalonic acid, which causes severe acidosis and also damages the central nervous system.7,8 In this study, we determined the crystal structure of TTE0360 from Thermoanaerobacter tengcongensis to 2.0 A resolution using single-wavelength anomalous dispersion (SAD) by short cryo-soaking with a high concentration of potassium iodide.9,10 In GenBank database, the gene tte0360 is predicted to be glyoxalase I, a member of glyoxalase superfamily. However, the TTE0360 structure is very different than the released structures of glyoxalase I. A search for the 3D homologs using DALI11 shows that TTE0360 has significant structural similarity with the MMCE from Propionibacterium shermanii,12 which is also a member of glyoxalase superfamily. The fold type of the TTE0360 molecule belongs to the vicinal-oxygen-chelate superfamily (VOC).13 The similarities of structure folds, dimer formation, and active sites between TTE0360 and MMCE, suggest that the protein TTE0360 may be the MMCE from T. tengcongensis.

Crystal structure of the N-terminal domain of human CDC73 and its implications for the hyperparathyroidism-jaw tumor (HPT-JT) syndrome

CDC73/Parafibromin is a critical component of the Paf1 complex (PAF1C), which is involved in transcriptional elongation and histone modifications. Mutations of the human CDC73/HRPT2 gene are associated with hyperparathyroidism-jaw tumor (HPT-JT) syndrome, an autosomal dominant disorder. CDC73/parafibromin was initially recognized as a tumor suppressor by inhibiting cell proliferation via repression of cyclin D1 and c-myc genes. In recent years, it has also shown oncogenic features by activating the canonical Wnt/β-catenin signal pathway. Here, through limited proteolysis analysis, we demonstrate that the evolutionarily conserved human CDC73 N-terminal 111 residues form a globularly folded domain (hCDC73-NTD). We have determined a crystal structure of hCDC73-NTD at 1.02 Å resolution, which reveals a novel protein fold. CDC73-NTD contains an extended hydrophobic groove on its surface that may be important for its function. Most pathogenic CDC73 missense mutations associated with the HPT-JT syndrome are located in the region encoding CDC73-NTD. Our crystal and biochemical data indicate that most CDC73 missense mutations disrupt the folding of the hydrophobic core of hCDC73-NTD, while others such as the K34Q mutant reduce its thermostability. Overall, our results provide a solid structural basis for understanding the structure and function of CDC73 and its association with the HPT-JT syndrome and other diseases.

Crystal structure of Tflp: A ferredoxin-like metallo-beta-lactamase superfamily protein from Thermoanaerobacter tengcongensis

Ferredoxins are a group of proteins containing nonheme iron and acid-labile sulfur. They are ubiquitous electron transfer proteins participating in a wide variety of redox reactions. Many organisms express multiple ferredoxins.1 Three types of [Fe-S] clusters have been described so far in ferredoxins: [2Fe-2S], [3Fe-4S], and [4Fe-4S]. The structures of these [Fe-S] clusters have substantial inherent stability in anaerobic solution, but oxygen can convert the exposed [Fe-S] clusters to unstable forms that decompose rapidly. Because of the high affinity of iron for thiolate residue, cysteine residue is the most common ligand of the Fe atom, but some other amino acid residues including histidine, aspartate, serine, and even backbone amide have also been found to coordinate the iron.2 Despite the limited [Fe-S] cluster types, ferredoxins present largely diverse structural architectures.3 A group of metallo-b-lactamase superfamily (MBLs) proteins were found to be ferredoxins bearing [2Fe-2S] cluster, but their crystal structures have not been reported.4 Here we report the crystal structure of a ferredoxin-like protein from Thermoanaerobacter tengcongensis (Tflp). Although Tflp is considered as a member of MBLs based on its threedimensional structural similarity, its crystal structure represents some novel structural features. A novel structural motif comprising four antiparallel b strands is found and a di-Fe center is located in the conserved active site. Tflp is one of the 15 proteins in T. tengcongensis considered to be unique to thermophiles.5 The gene encoding Tflp clusters with another six genes encoding ferredoxins. The ultraviolet/visible (UV/Vis) and electronic paramagnetic resonance (EPR) spectra reveal the existence of a [Fe-S] cluster in Tflp reconstituted with sulfur and iron under dithionite-reduced condition. These results characterize Tflp as a kind of ferredoxin.