Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5

Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.     

Directed evolution of a highly active Yersinia mollaretii phytase

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073?U/mg which is ～10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ～8,400 clones) a phytase variant with ～20% improved thermostability (58°C for 20?min; residual activity wild type ～34%; variant ～53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993?U/mg).     

Induction of alkane hydroxylase proteins by unoxidized alkane in Pseudomonas putida.

In vitro complementation assays have been used to demonstrate the induction of alkane hydroxylase proteins in mutants lacking the ability to convert n-alkanes to their primary alcohols. Purified heptane is an effective inducer in a mutant lacking detectable hydroxylase activity.     

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.     

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.     

Topology of the membrane-bound alkane hydroxylase of Pseudomonas oleovorans.

The Pseudomonas oleovorans alkane hydroxylase is an integral cytoplasmic membrane protein that is expressed and active in both Escherichia coli and P. oleovorans. Its primary sequence contains eight hydrophobic stretches that could span the membrane as alpha-helices. The topology of alkane hydroxylase was studied in E. coli using protein fusions linking different amino-terminal fragments of the alkane hydroxylase (AlkB) to alkaline phosphatase (PhoA) and to beta-galactosidase (LacZ). Four AlkB-PhoA fusions were constructed using transposon TnphoA. Site-directed mutagenesis was used to create PstI sites at 12 positions in AlkB. These sites were used to create AlkB-PhoA and AlkB-LacZ fusions. With respect to alkaline phosphatase and beta-galactosidase activity each set of AlkB-PhoA and AlkB-LacZ fusions revealed the expected complementary activities. At three positions, PhoA fusions were highly active, whereas the corresponding LacZ fusions were the least active. At all other positions the PhoA fusions were almost completely inactive, but the corresponding LacZ fusions were highly active. These data predict a model for alkane hydroxylase containing six transmembrane segments. In this model the amino terminus, two hydrophilic loops, and a large carboxyl-terminal domain are located in the cytoplasm. Only three very short loops near amino acid positions 52, 112, and 251 are exposed to the periplasm.     

Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase

LadA, a long-chain alkane monooxygenase, utilizes a terminal oxidation pathway for the conversion of long-chain alkanes (up to at least C₃₆) to corresponding primary alcohols in thermophilic bacillus Geobacillus thermodenitrificans NG80-2. Here, we report the first structure of the long-chain alkane hydroxylase, LadA, and its complex with the flavin mononucleotide (FMN) coenzyme. LadA is characterized as a new member of the SsuD subfamily of the bacterial luciferase family via a surprising structural relationship. The LadA:FMN binary complex structure and a LadA:FMN:alkane model reveal a hydrophobic cavity that has dual roles: to provide a hydrogen-bond donor (His138) for catalysis and to create a solvent-free environment in which to stabilize the C4a-hydroperoxyflavin intermediate. Consequently, LadA should catalyze the conversion of long-chain alkanes via the acknowledged flavoprotein monooxygenase mechanism. This finding suggests that the ability of LadA to catalyze the degradation of long-chain alkanes is determined by the binding mode of the long-chain alkane substrates. The LadA structure opens a rational perspective to explore and alter the substrate binding site of LadA, with potential biotechnological applications in areas such as petroleum exploration and treatment of environmental oil pollution.     

A highly active, soluble mutant of the membrane-associated (S)-mandelate dehydrogenase from Pseudomonas putida.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry 32, 12959-12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH-GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH-GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, approximately 39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH-GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH-GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.     

Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR

AIMS: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. METHODS AND RESULTS: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (相似文献   

Crystallization of a soluble,catalytically active form of Escherichia coli leader peptidase

Leader peptidase, a novel serine protease in Escherichia coli, catalyzes the cleavage of the amino-terminal leader sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Here, we report a procedure for the purification and the crystallization of a soluble non-membrane-bound form of leader peptidase (Δ2-75). Crystals were obtained by the sitting-drop vapor diffusion technique using ammonium dihydrogen phosphate as the precipitant. Interestingly, we have found that the presence of the detergent Triton X-100 is required to obtain crystals sufficiently large for X-ray analysis. The crystals belong to the tetragonal space group P4₂2₁2, with unit cell dimensions of a = b = 115 Å and c = 100 Å, and contain 2 molecules per asymmetric unit. This is the first report of the crystallization of a leader (or signal) peptidase. © 1995 Wiley-Liss, Inc.     

Isolation of a highly active preparation of beta-D-galactosidase

Methods for isolation and purification of beta-galactosidase from Bacillus subtilis, st. IBP-101 are described. The bacterial cells were disrupted by different procedures such as freezing and thawing with subsequent autolysis at 37 degrees C, disrupting in a French press DKM-3 or in ultrasonic disintegrators UZDN-1 (USSR) and Soniprep-150. It is shown that the specific activity and yield of the enzyme depends to a great extent on the disrupting procedure used. The best results were obtained in case of sonication. The preparation was purified by precipitation with ammonium sulphate (25-75% saturated) and chromatography on DEAE-cellulose and DEAE-Sephadex. The purified enzyme had a specific activity of 3155 units per mg protein. The molecular weight of the homogeneous according to gel polyacrylamide electrophoresis preparation was 215,000, as estimated by gel filtration, and 105,000, as estimated by SDS gel electrophoresis. The enzyme retains the activity in the presence of Na+, Mn2+ or Mg2+ ions or the thiolic reagents, dithiothreitol or 2-mercaptoethanol. The pH optimum of the enzyme activity is 6.3 and it is stable in water solutions at pH from 6 to 9 and can be lyophilized. The given preparation of beta-galactosidase has a high affinity for synthetic substrates such as o- and p-nitrophenyl-beta-D-galactopyranosides and 4-methylumbelliferyl-beta-D-galactopyranoside.     

Generation of a complete, soluble, and catalytically active sterol 14 alpha-demethylase-reductase complex.

Sterol 14 alpha-demethylation is one of the key steps of sterol biosynthesis in eukaryotes and is catalyzed by cytochrome P450 sterol 14 alpha-demethylase (other names being CYP51 and P45014DM) encoded by ERG11. This enzyme activity is supported by an associated NAPDH-dependent reductase encoded by NCPR1 (NCP1), which is also associated with the endoplasmic reticulum. A diglycine linker recognition site (Gly-Gly-Ile-Glu-Gly-Arg-Gly-Gly) for the protease factor Xa, also containing a thrombin recognition site, was inserted just beyond the N-terminal hydrophobic segment of Candida albicans Erg11p. This modified enzyme was heterologously expressed at a level of 2.5 nmol of Erg11p/mg of protein as an integral endoplasmic reticulum protein. Following purification, treatment of the modified protein with factor Xa or thrombin resulted in sequence-specific cleavage and production of a soluble N-terminal truncated Erg11p which exhibited spectral characteristics identical to those of the purified full-length, wild-type form. Furthermore, reconstitution of the soluble enzyme with soluble yeast Ncpr1p, expressed and purified as an N-terminal deletion of 33 amino acids encompassing its membrane anchor, resulted in a fully functional and soluble eukaryotic Erg11p system. The complex was disrupted by high-salt concentration, reflecting the importance of electrostatic forces in the protein-protein interaction. The results demonstrate the membrane anchor serves to localize Erg11p to the ER where the substrate is located, but is not essential in either Ncpr1p or Erg11p activity. The possibility of cocrystallization of an active soluble eukaryotic 14 alpha-demethylase can be envisaged.     

Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase

The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution.