Extra- and Intracellular Imaging of Human Matrix Metalloprotease 11 (hMMP-11) with a Cell-penetrating FRET Substrate

Matrix metalloprotease 11 (MMP-11), a protease associated with invasion and aggressiveness of cancerous tissue, was postulated as a prognostic marker for pancreatic, breast, and colon cancer patients. Expression analysis, however, did not reveal localization and regulation of this protease. Thus, cellular tools for the visualization of MMP-11 are highly desirable to monitor presence and activity and to elucidate the functional role of MMP-11. Therefore, fluorescein-Dabcyl-labeled Foerster resonance energy transfer (FRET) substrates were developed. The design focused on enhanced peptide binding to human MMP-11, employing an unusual amino acid for the specificity pocket P1′. The addition of several arginines resulted in a cell-permeable FRET substrate SM-P124 (Ac-GRRRK(Dabcyl)-GGAANC(MeOBn)RMGG-fluorescein). In vitro evaluation of SM-P124 with human MMP-11 showed a 25-fold increase of affinity (k_cat/K_m = 9.16 × 10³ m⁻¹ s⁻¹, K_m = 8 μm) compared with previously published substrates. Incubation of pancreatic adenocarcinoma cell line MIA PaCa-2 and mamma adenocarcinoma cell line MCF-7 with the substrate SM-P124 (5 μm) indicated intra- and extracellular MMP-11 activity. A negative control cell line (Jurkat) showed no fluorescent signal either intra- or extracellularly. Negative control FRET substrate SM-P123 produced only insignificant extracellular fluorescence without any intracellular fluorescence. SM-P124 therefore enabled intra- and extracellular tracking of MMP-11-overexpressing cancers such as pancreatic and breast adenocarcinoma and might contribute to the understanding of the activation pathways leading to MMP-11-mediated invasive processes.     

Biochemical Characterization of a Dihydroneopterin Aldolase Used for Methanopterin Biosynthesis in Methanogens

The gene encoding 7,8-dihydroneopterin aldolase (DHNA) was recently identified in archaea through comparative genomics as being involved in methanopterin biosynthesis (V. Crécy-Lagard, G. Phillips, L. L. Grochowski, B. El Yacoubi, F. Jenney, M. W. Adams, A. G. Murzin, and R. H. White, ACS Chem. Biol. 7:1807–1816, 2012, doi:10.1021/cb300342u). Archaeal DHNA shows a unique secondary and quaternary structure compared with bacterial and plant DHNAs. Here, we report a detailed biochemical examination of DHNA from the methanogen Methanocaldococcus jannaschii. Kinetic studies show that M. jannaschii DHNA possesses a catalytic capability with a k_cat/K_m above 10⁵ M⁻¹ s⁻¹ at 70°C, and at room temperature it exhibits a turnover number (0.07 s⁻¹) comparable to bacterial DHNAs. We also found that this enzyme follows an acid-base catalytic mechanism similar to the bacterial DHNAs, except when using alternative catalytic residues. We propose that in the absence of lysine, which is considered to be the general base in bacterial DHNAs, an invariant water molecule likely functions as the catalytic base, and the strictly conserved His35 and Gln61 residues serve as the hydrogen bond partners to adjust the basicity of the water molecule. Indeed, substitution of either His35 or Gln61 causes a 20-fold decrease in k_cat. An invariant Tyr78 is also shown to be important for catalysis, likely functioning as a general acid. Glu25 plays an important role in substrate binding, since replacing Glu25 by Gln caused a ≥25-fold increase in K_m. These results provide important insights into the catalytic mechanism of archaeal DHNAs.     

A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase

Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3′-azido-2′,3′-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5′-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s⁻¹) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 µM) and chemistry forward and reverse reactions, 0.38 and 0.22 s⁻¹, respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing k_cat/K_m (7 × 10⁻⁶ μ M⁻¹ s⁻¹). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5′-triphosphate (8-oxo-dGTP) show this same phenomena.     

Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T _m) and the urea midtransition concentration (C _m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T _m and C _m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.     

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K_m = 7.9 mM; k_cat/K_m = 0.73 mM⁻¹ s⁻¹). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k_cat/K_m = 450 mM⁻¹ s⁻¹) than with NADPH (k_cat/K_m = 5.5 mM⁻¹ s⁻¹), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu²⁺ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase: STRUCTURE OF THE ENZYME·PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C–H BOND CLEAVAGE*

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both k_cat and k_cat/K_m, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (k_on 2.4 × 10⁷ m⁻¹ s⁻¹) is only ∼2-fold greater than the catalytic efficiency (k_cat/K_m = 1.3 × 10⁷ m⁻¹ s⁻¹) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with k_cat ≈ 8 x 10⁻³ s⁻¹ and K_M < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower k_cat and a K_M ≈ 300 nM. The rate of ligation increased with addition of Mn²⁺, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Streptomyces coelicolor Polynucleotide Phosphorylase Can Polymerize Nucleoside Diphosphates under Phosphorolysis Conditions,with Implications for the Degradation of Structured RNAs

We have examined the ability of wild-type polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor and two mutant forms of the enzyme, N459D and C468A, to function in the polymerization of ADP and in the phosphorolysis of RNA substrates derived from the S. coelicolor rpsO-pnp operon. The wild-type enzyme was twice as active in polymerization as N459D and four times as active as C468A. The k_cat/K_m value for phosphorolysis of a structured RNA substrate by N459D was essentially the same as that observed for the wild-type enzyme, while C468A was 50% as active with this substrate. A mixture of all four common nucleoside diphosphates increased the k_cat/K_m for phosphorolysis of the structured substrate by the wild-type enzyme by a factor of 1.7 but did not affect phosphorolysis catalyzed by N459D or C468A. We conducted phosphorolysis of the structured substrate in the presence of nucleoside diphosphates and labeled the 3′ ends of the products of those reactions using [³²P]pCp. Digestion of the end-labeled RNAs and display of the products on a sequencing gel revealed that wild-type S. coelicolor PNPase was able to synthesize RNA 3′ tails under phosphorolysis conditions while the N459D and C468A mutants could not. The wild-type enzyme did not add 3′ tails to a substrate that already possessed an unstructured 3′ tail. We propose a model in which the transient synthesis of 3′ tails facilitates the phosphorolysis of structured substrates by Streptomyces PNPase.     

OptZyme: Computational Enzyme Redesign Using Transition State Analogues

OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., k_cat or k_cat/K_M) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that K_M correlates (R² = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, k_cat/K_M correlates (R² = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and k_cat correlates (R² = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved K_M, k_cat, and k_cat/K_M for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving K_M, k_cat, or k_cat/K_M. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S.     

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k_cat/K_m = 10⁵-10⁷ m⁻¹ s⁻¹). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.     

Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay

Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5′-fluorescein-labeled RNA hybridized to a complementary 3′-quencher-modified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a k_cat/K_m of 330 000 M⁻¹ s⁻¹, a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function.     

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k_unw = 5.5 s⁻¹ and a dissociation step from partially unwound DNA of k_off = 1.9 s⁻¹. These results indicate a kinetic step size of about 3–4 bp, unwinding rate of about 15–20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5′-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.     

Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD⁺ salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (k_cat/K_m = 43.5 mM⁻¹s⁻¹) than pyrazinamidase (k_cat/K_m = 3.2 mM⁻¹s⁻¹). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.     

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_cat/K_m = 1000 ± 100 m⁻¹ s⁻¹ versus 700 ± 100 m⁻¹ s⁻¹). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_cat/K_m = 80 ± 40 m⁻¹ s⁻¹). In the presence of 3-HSA the K_mapp for O₂ was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme''s substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val³⁶⁷–Val³⁹⁴) could adopt two conformations differing by a rigid body rotation of 25° around Arg³⁶⁶. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme''s substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.     

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6¹,6³- and 6¹,6⁴-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the k_cat and K_m values for transglycosylation were 1.78 × 10³ s⁻¹ and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10³ s⁻¹ and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the k_cat/K_m ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.     

Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA)

Escherichia coli DNA ligase (EcoLigA) repairs 3′-OH/5′-PO₄ nicks in duplex DNA via reaction of LigA with NAD⁺ to form a covalent LigA-(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the nick 5′-PO₄ to form an AppDNA intermediate (step 2); and attack of the nick 3′-OH on AppDNA to form a 3′-5′ phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA–AMP. For substrates with correctly base-paired 3′-OH/5′-PO₄ nicks, k_step2 was fast (6.8–27 s⁻¹) and similar to k_step3 (8.3–42 s⁻¹). Absent ammonium, k_step2 and k_step3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3′-OH base mispairs and 3′ N:abasic lesions, which elicited 1000- to >20000-fold decrements in k_step2. The exception was the non-canonical 3′ A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3′ nucleoside for catalysis of 5′ adenylylation; and (ii) EcoLigA''s potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5′-phosphate base mispairs and 5′ N:abasic lesions.