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ABSTRACT In multi-domain proteins, the domains typically run end-to-end, that is, one domain follows the C-terminus of another domain. However, approximately 10% of multi-domain proteins are formed by insertion of one domain sequence into that of another domain. Detecting such insertions within protein sequences is a fundamental challenge in structural biology. The haloacid dehalogenase superfamily (HADSF) serves as a challenging model system wherein a variable cap domain (∼5-200 residues in length) accessorizes the ubiquitous Rossmann-fold core domain, with variations in insertion site and topology corresponding to different classes of cap types. Herein, we describe a comprehensive computational strategy, CapPredictor, for determining large, variable domain insertions in protein sequences. Using a novel sequence-alignment algorithm in conjunction with a structure-guided sequence profile from 154 core-domain-only structures, more than 40,000 HADSF member sequences were assigned cap types. The resulting data set afforded insight into HADSF evolution. Notably, a similar distribution of cap-type classes across different phyla was observed, indicating that all cap types existed in the last universal common ancestor. In addition, comparative analyses of the predicted cap-type and functional assignments showed that different cap types carry out similar chemistries. Thus, while cap domains play a role in substrate recognition and chemical reactivity, cap-type does not strictly define functional class. Through this example, we have shown that CapPredictor is an effective new tool for the study of form and function in protein families where domain insertion occurs. Proteins 2014; 82:1896-1906. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Highlights: [•] Facultative anaerobes of the phyla Bacteroidetes synthesize menaquinone. [•] The pathway 1,4-dihydroxnapthoyl-CoA thioesterase is a type AB hotdog-fold protein. [•] In some species the thioesterase is fused to one of two copies of a HAD phosphatase. [•] The HAD phosphatase domain functions in the oligomerization of the thioesterase domain. [•] As a result of the fusion event the HAD phosphatase domain lost catalytic activity. [ABSTRACT FROM AUTHOR]

Analysis of the haloalkanoate dehalogenase superfamily (HADSF) has uncovered homologues occurring within the same organism that are found to possess broad, overlapping substrate specificities, and low catalytic efficiencies. Here we compare the HADSF phosphatase BT1666 from Bacteroides thetaiotaomicron VPI-5482 to a homologue with high sequence identity (40%) from the same organism BT4131, a known hexose-phosphate phosphatase. The goal is to find whether these enzymes represent duplicated versus paralogous activities. The X-ray crystal structure of BT1666 was determined to 1.82 Å resolution. Superposition of the BT1666 and BT4131 structures revealed a conserved fold and identical active sites suggestive of a common physiological substrate. The steady-state kinetic constants for BT1666 were determined for a diverse panel of phosphorylated metabolites to define its substrate specificity profile and overall level of catalytic efficiency. Whereas BT1666 and BT4131 are both promiscuous, their substrate specificity profiles are distinct. The catalytic efficiency of BT1666 ( kcat/ Km = 4.4 × 102 M−1 s−1 for the best substrate fructose 1,6-(bis)phosphate) is an order of magnitude less than that of BT4131 ( kcat/ Km = 6.7 × 103 M−1 s−1 for 2-deoxyglucose 6-phosphate). The seemingly identical active-site structures point to sequence variation outside the active site causing differences in conformational dynamics or subtle catalytic positioning effects that drive the divergence in catalytic efficiency and selectivity. The overlapping substrate profiles may be understood in terms of differential regulation of expression of the two enzymes or a conferred advantage in metabolic housekeeping functions by having a larger range of possible metabolites as substrates. Proteins 2011;. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Carbamate kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP and ammonium carbamate, which is hydrolyzed to ammonia and carbonate. The three-dimensional structure of carbamate kinase from the human parasite Giardia lamblia ( glCK) has been determined at 3 Å resolution. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 69.77, b = 85.41, c = 102.1 Å, β = 106.8°. The structure was refined to a final R factor of 0.227. The essentiality of glCK together with its absence in humans makes the enzyme an attractive candidate for anti- Giardia drug development. Steady-state kinetic rate constants have been determined. The kcat for ATP formation is 319 ± 9 s−1. The Km values for carbamoyl phosphate and ADP are 85 ± 6 and 70 ± 5 µ M, respectively. The structure suggests that three invariant lysine residues (Lys131, Lys216 and Lys278) may be involved in the binding of substrates and phosphoryl transfer. The structure of glCK reveals that a glycerol molecule binds in the likely carbamoyl phosphate-binding site. [ABSTRACT FROM AUTHOR]

Aspergillus niger produces oxalic acid through the hydrolysis of oxaloacetate, catalyzed by the cytoplasmic enzyme oxaloacetate acetylhydrolase (OAH). The A. niger genome encodes four additional open reading frames with strong sequence similarity to OAH yet only the oahA gene encodes OAH activity. OAH and OAH-like proteins form subclass of the isocitrate lyase/PEP mutase enzyme superfamily, which is ubiquitous present filamentous fungi. Analysis of function-specific residues using a superfamily-based approach revealed an active site serine as a possible sequence marker for OAH activity. We propose that presence of this serine in family members correlates with presence of OAH activity whereas its absence correlates with absence of OAH. This hypothesis was tested by carrying out a serine mutagenesis study with the OAH from the fungal oxalic acid producer Botrytis cinerea and the OAH active plant petal death protein as test systems. Proteins 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO_2114, from the plant pathogen Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO_2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO_2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO_2114 was confirmed by kinetic assays. To explore PSPTO_2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO_2114 homologs were mapped onto the PSPTO_2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst. Proteins 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Phosphonoacetaldehyde hydrolase, a C-P bond-cleaving enzyme which utilizes an unusual bicovalent catalytic strategy, has been crystallized by the hanging-drop vapor-diffusion method using PEG 4000 as the precipitant. The crystals belong to the monoclinic system and belong to space group C2, with unit-cell parameters a = 210.5, b = 45.5, c = 64.7 Å, β = 105.0°. The asymmetric unit contains a dimer related by a non-crystallographic dyad In addition to a 27 Å native data set, the following data sets have been collected: a 2.4 Å data set from crystals complexed with the intermediate analog vinyl sulfonate, a 3.0 Å three-wavelength MAD data set from crystals complexed with the product analog WO42-, as well as several heavy-atom data sets to 3.0 Å or better, of which only three have proven useful for MIR calculations. Examination of the native Patterson map revealed NCS that made previously uninterpretable derivative data useful. Independent phase sets were first calculated and refined for the MAD and MIR experiments separately and were then combined. The combined phase set was further improved by solvent flattening, histogram matching and NCS averaging. Interpretation of the resulting electron-density map is currently under way. [ABSTRACT FROM AUTHOR]

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol-pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl-CoA and acyl-N-acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl-CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not. [Copyright &y& Elsevier]

β-Phosphoglucomutase (β-PGM), a 28 kDa monomer, catalyzes the reversible conversion of β-D-glucose-1-phosphate to β-D-glucose- 6-phosphate in maltose metabolism in a variety of organisms. Sequence analysis of β-PGM indicates that it is a member of the halo acid dehalogenase (HAD) enzyme superfamily, which evolved to cleave C-Cl, C-P and C-OP bonds in a variety of substrates. β-PGM has been crystallized using the hanging-drop method. Diffraction-quality crystals of the native protein have been obtained from two conditions, both belonging to space group P212121, with unit-cell parameters a = 53.67, b = 92.78, c = 111.60 and a = 53.21, b = 57.01, c = 76.11 Å. To solve the phase problem, selenomethionine (SeMet) containing β-PGM crystals have been grown. The SeMet-containing crystals diffract to high resolution only when grown by microseeding with native crystals. A three-wavelength data set has been collected to 2.3 Å on crystals of the SeMet-substituted β-PGM. The structure solution is currently being attempted by the multi-wavelength anomalous diffraction (MAD) phasing method. [ABSTRACT FROM AUTHOR]

The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0 mm were obtained in space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7 Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18 d. Initial refinement to 2.3 Å resolution using only neutron data showed significant density for catalytically important residues.

Beta-phosphoglucomutase (beta-PGM), a 28 kDa monomer, catalyzes the reversible conversion of beta-D-glucose-1-phosphate to beta-D-glucose-6-phosphate in maltose metabolism in a variety of organisms. Sequence analysis of beta-PGM indicates that it is a member of the haloacid dehalogenase (HAD) enzyme superfamily, which evolved to cleave C-Cl, C-P and C-OP bonds in a variety of substrates. beta-PGM has been crystallized using the hanging-drop method. Diffraction-quality crystals of the native protein have been obtained from two conditions, both belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.67, b = 92.78, c = 111.60 and a = 53.21, b = 57.01, c = 76.11 A. To solve the phase problem, selenomethionine (SeMet) containing beta-PGM crystals have been grown. The SeMet-containing crystals diffract to high resolution only when grown by microseeding with native crystals. A three-wavelength data set has been collected to 2.3 A on crystals of the SeMet-substituted beta-PGM. The structure solution is currently being attempted by the multiwavelength anomalous diffraction (MAD) phasing method.