The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni.

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDOKF1 from Comamonas testosteroni KF1 and found that it had an apparent kcat/Km for phthalate of 0.58 ± 0.09 µM-1s-1, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDOKF1 larger than that of other characterized ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

High expression of ring-hydroxylating dioxygenase genes ensure efficient degradation of p-toluate, phthalate, and terephthalate by Comamonas testosteroni strain 3a2.

Para-toluic acid, a major pollutant in industrial wastewater, is hazardous to human health. It has been demonstrated that Gram-negative bacteria are among the most effective degraders of para-toluic acid. In this study, the ability of Comamonas testosteroni strain 3a2, isolated from a petrochemical industry wastewater, to degrade para-toluic acid was investigated. The effect of different carbon (glucose and ethylene glycol) and nitrogen sources (urea, yeast extract, peptone, NaNO3, NH4NO3) on the biodegradation of para-toluic acid by the isolate 3a2 was evaluated. Furthermore, ring hydroxylating dioxygenase genes were amplified by PCR and their expression was evaluated during the biodegradation of para-toluic acid. The results indicated that strain 3a2 was able to degrade up to 1000 mg/L of para-toluic acid after 14 h. The highest degradation yield was recorded in the presence of yeast extract as nitrogen source. However, the formation of terephthalic acid and phthalic acid was noted during para-toluic acid degradation by the isolate 3a2. Toluate 1,2-dioxygenase, terephthalate 1,2 dioxygenase, and phthalate 4,5 dioxygenase genes were detected in the genomic DNA of 3a2. The induction of ring hydroxylating dioxygenase genes was proportional to the concentration of each hydrocarbon. This study showed that the isolate 3a2 can produce terephthalate and phthalate during the para-toluic acid biodegradation, which were also degraded after 24 h.

Characterization of a LuxR repressor for 3,17ß-HSD in Comamonas testosteroni ATCC11996.

3,17ß-Hydroxysteroid dehydrogenase in Comamonas testosteroni (C. testosteroni) is a key enzyme involved in the degradation of steroid compounds. Recently, we found that LuxR is a negative regulator in the expression of the 3,17ß-HSD gene. In the present work, we cultured wild-type and LuxR knock-out mutants of C. testosteroni with inducers such as testosterone, estradiol, progesterone or estrone. HPLC analysis showed that the degradation activities towards testosterone, estradiol, progesterone, and estrone by C.T.-LuxR-KO1 were increased by 7.1%, 9.7%, 11.9% and 3.1%, respectively compared to the wild-type strain. Protein conformation of LuxR was predicted by Phyre 2 Server software, where the N-terminal 86(Ile), 116(Ile), 118(Met) and 149(Phe) residues form a testosterone binding hydrophobic pore, while the C-terminus forms the DNA binding site (HTH). Further, luxr point mutant plasmids were prepared by PCR and co-transformed with pUC3.2-4 into E. coli HB101. ELISA was used to determine 3,17ß-HSD expression after testosterone induction. Compared to wild-type luxr, 3,17ß-HSD expression in mutants of I86T, I116T, M118T and F149S were decreased. The result indicates that testosterone lost its capability to bind to LuxR after the four amino acid residues had been exchanged. No significant changes of 3,17ß-HSD expression were found in K354I and Y356 N mutants compared to wild-type luxr, which indicates that these two amino acid residues in LuxR might relate to DNA binding. Native LuxR protein was prepared from inclusion bodies using sodium lauroylsarcosinate. Molecular interaction experiments showed that LuxR protein binds to a nucleotide sequence which locates 87 bp upstream of the ßhsd promoter. Our results revealed that steroid induction of 3,17ß-HSD in C. testosteroni in fact appears to be a de-repression, where testosterone prevents the LuxR regulator protein binding to the 3,17ß-HSD promoter domain.

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta-gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

Efficient heterologous expression of nicotinate dehydrogenase in Comamonas testosteroni CNB-2 with transcriptional, folding enhancement strategy.

Nicotinate dehydrogenase (NDHase) from Comamonas testosteroni JA1 catalyzes the C6 hydroxylation of 3-cyanopyridine with high regional selectivity, which is a very difficult and complex reaction for chemical synthesis. However, because NDHase is a membrane protein with three subunits (ndhS, ndhL and ndhM), it is difficult to express the enzyme in a functional form using common hosts such as Escherichia coli, Bacilus subtilis or Pichia pastoris. Furthermore, the enzyme requires special electron transfer chains in the membrane system for proper catalytic activity. Thus, we investigated the expression of NDHase in non-model bacterial strains, which are evolutionarily similar to C. testosteroni JA1, using several broad-host plasmids with different copy numbers as expression vectors. We successfully expressed NDHase in soluble from using the pVLT33 vector in C. testosteroni CNB-2, and found the activity of enzyme to be 40.6 U/L. To further improve the expression of NDHase in C. testosteroni CNB-2, we trialed a T7-like MmP1 system, composed of MmP1 RNA polymerase and an MmP1 promoter, which is used for transcriptional control in non-model bacteria. This increased protein expression and enzyme activity doubled to 90.5 U/L. A molecular chaperone was co-expressed using pBBR1 MCS-5 in the same host to improve the efficiency of folding and assembly of multi-subunit structures. The maximum activity was 115 U/L using the molecular chaperone GroES-EL, far surpassing the previously reported level, although expression was almost equivalent. These results indicate that a strategy involving the construction of a T7-like system and co-expression of a molecular chaperone offers an efficient approach for heterologous expression of enzymes that are difficult to express in functional forms using conventional hosts.

Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire ß-Oxidation Cycle of the Cleaved B Ring.

Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by ß-oxidation. In this study, we revealed that 7ß,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of ß-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7ß,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters.IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire ß-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

Steroid Degradation in Comamonas testosteroni TA441: Identification of Metabolites and the Genes Involved in the Reactions Necessary before D-Ring Cleavage.

Bacterial steroid degradation has been studied mainly with Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni as representative steroid degradation bacteria for more than 50 years. The primary purpose was to obtain materials for steroid drugs, but recent studies showed that many genera of bacteria (Mycobacterium, Rhodococcus, Pseudomonas, etc.) degrade steroids and that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment. The role of bacterial steroid degradation in the environment is, however, yet to be revealed. To uncover the whole steroid degradation process in a representative steroid-degrading bacterium, C. testosteroni, to provide basic information for further studies on the role of bacterial steroid degradation, we elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage in C. testosteroni TA441. In bacterial oxidative steroid degradation, A- and B-rings of steroids are cleaved to produce 2-hydroxyhexa-2,4-dienoic acid and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. The latter compound was revealed to be degraded to the coenzyme A (CoA) ester of 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid, which is converted to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid by ORF31-encoded hydroxylacyl dehydrogenase (ScdG), followed by conversion to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid by ORF4-encoded acyl-CoA dehydrogenase (ScdK). Then, a water molecule is added by the ORF5-encoded enoyl-CoA hydratase (ScdY), which leads to the cleavage of the D-ring. The conversion by ScdG is presumed to be a reversible reaction. The elucidated pathway in C. testosteroni TA441 is different from the corresponding pathways in Mycobacterium tuberculosis H37Rv.IMPORTANCE Studies on representative steroid degradation bacteria Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni were initiated more than 50 years ago primarily to obtain materials for steroid drugs. A recent study showed that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment, but the role of bacterial steroid degradation in the environment is yet to be revealed. This study aimed to uncover the whole steroid degradation process in C. testosteroni TA441, in which major enzymes for steroidal A- and B-ring cleavage were elucidated, to provide basic information for further studies on bacterial steroid degradation. C. testosteroni is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption. We elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage, which appeared to differ from those present in Mycobacterium tuberculosis H37Rv.

Dynamical origins of heat capacity changes in enzyme-catalysed reactions.

Heat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed, along with changes in energetic fluctuations across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.

An Activator-Blocker Pair Provides a Controllable On-Off Switch for a Ketosteroid Isomerase Active Site Mutant.

Control of enzyme activity is fundamental to biology and represents a long-term goal in bioengineering and precision therapeutics. While several powerful molecular strategies have been developed, limitations remain in their generalizability and dynamic range. We demonstrate a control mechanism via separate small molecules that turn on the enzyme (activator) and turn off the activation (blocker). We show that a pocket created near the active site base of the enzyme ketosteriod isomerase (KSI) allows efficient and saturable base rescue when the enzyme's natural general base is removed. Binding a small molecule with similar properties but lacking general-base capability in this pocket shuts off rescue. The ability of small molecules to directly participate in and directly block catalysis may afford a broad controllable dynamic range. This approach may be amenable to numerous enzymes and to engineering and screening approaches to identify activators and blockers with strong, specific binding for engineering and therapeutic applications.

Functional analysis of a novel repressor LuxR in Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) ATCC11996 is a gram negative bacterium which can use steroid as a carbon and energy source. 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme for the degradation of steroid hormones in C. testosteroni. The LuxR regulation family is a group of regulatory proteins which play important role in gram negative bacterium. The luxr gene is located on 58 kb upstream of 3,17ß-HSD gene with the opposite transcription orientation in the chromosomal DNA of C. testosteroni. An open reading frame of this putative luxr gene consists of 1125 bp and is translated into a protein containing 374 amino acids. The luxr gene was cloned into plasmid pK18 and plasmid pK-LuxR1 was obtained. E. coli HB101 was co-transformed by pK-LuxR1 and pUC912-10, pUC1128-5 or pUC3.2-4 (which contain ßhsd gene and different length promoter, repeat sequences). The result of ELISA showed that LuxR protein is a negative regulator for 3,17ß-HSD expression. The luxr gene in C. testosteroni was knock-out by homologous integration. 3,17ß-HSD expression was increased in the mutant (C.T.-L-KO1) comparing to that in wild-type C. testosteroni (C.T.) after 0.5 mM testosterone induction. The mutant C.T.-L-KO1 and wild-type C. testosteroni were cultured at 27 °C and 37 °C. The result of growth curve proved that LuxR has also effect on the bacterial growth.

Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3α-hydroxysteroid dehydrogenase/carbonyl reductase.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in kcat, and 37-, 1.9 × 106-, and 1.8 × 106-fold in kcat/KB, respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD+ based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The kcat/KB value results in ΔG‡ of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in kcat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis, resulting in the increases in kcat and kcat/KB.

Kemp Eliminase Activity of Ketosteroid Isomerase.

Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

Engineering Mycobacterium smegmatis for testosterone production.

A new biotechnological process for the production of testosterone (TS) has been developed to turn the model strain Mycobacterium smegmatis suitable for TS production to compete with the current chemical synthesis procedures. We have cloned and overexpressed two genes encoding microbial 17ß-hydroxysteroid: NADP 17-oxidoreductase, from the bacterium Comamonas testosteroni and from the fungus Cochliobolus lunatus. The host strains were M. smegmatis wild type and a genetic engineered androst-4-ene-3,17-dione (AD) producing mutant. The performances of the four recombinant bacterial strains have been tested both in growing and resting-cell conditions using natural sterols and AD as substrates respectively. These strains were able to produce TS from sterols or AD with high yields. This work represents a proof of concept of the possibilities that offers this model bacterium for the production of pharmaceutical steroids using metabolic engineering approaches.

Identification and characterization of two new 5-keto-4-deoxy-D-Glucarate Dehydratases/Decarboxylases.

BACKGROUND: Hexuronic acids such as D-galacturonic acid and D-glucuronic acid can be utilized via different pathways within the metabolism of microorganisms. One representative, the oxidative pathway, generates α-keto-glutarate as the direct link entering towards the citric acid cycle. The penultimate enzyme, keto-deoxy glucarate dehydratase/decarboxylase, catalyses the dehydration and decarboxylation of keto-deoxy glucarate to α-keto-glutarate semialdehyde. This enzymatic reaction can be tracked continuously by applying a pH-shift assay. RESULTS: Two new keto-deoxy glucarate dehydratases/decarboxylases (EC 4.2.1.41) from Comamonas testosteroni KF-1 and Polaromonas naphthalenivorans CJ2 were identified and expressed in an active form using Escherichia coli ArcticExpress(DE3). Subsequent characterization concerning K m, k cat and thermal stability was conducted in comparison with the known keto-deoxy glucarate dehydratase/decarboxylase from Acinetobacter baylyi ADP1. The kinetic constants determined for A. baylyi were K m 1.0 mM, k cat 4.5 s-1, for C. testosteroni K m 1.1 mM, k cat 3.1 s-1, and for P. naphthalenivorans K m 1.1 mM, k cat 1.7 s-1. The two new enzymes had a slightly lower catalytic activity (increased K m and a decreased k cat) but showed a higher thermal stability than that of A. baylyi. The developed pH-shift assay, using potassium phosphate and bromothymol blue as the pH indicator, enables a direct measurement. The use of crude extracts did not interfere with the assay and was tested for wild-type landscapes for all three enzymes. CONCLUSIONS: By establishing a pH-shift assay, an easy measurement method for keto-deoxy glucarate dehydratase/decarboxylase could be developed. It can be used for measurements of the purified enzymes or using crude extracts. Therefore, it is especially suitable as the method of choice within an engineering approach for further optimization of these enzymes.

Evaluation of the Catalytic Contribution from a Positioned General Base in Ketosteroid Isomerase.

Proton transfer reactions are ubiquitous in enzymes and utilize active site residues as general acids and bases. Crystal structures and site-directed mutagenesis are routinely used to identify these residues, but assessment of their catalytic contribution remains a major challenge. In principle, effective molarity measurements, in which exogenous acids/bases rescue the reaction in mutants lacking these residues, can estimate these catalytic contributions. However, these exogenous moieties can be restricted in reactivity by steric hindrance or enhanced by binding interactions with nearby residues, thereby resulting in over- or underestimation of the catalytic contribution, respectively. With these challenges in mind, we investigated the catalytic contribution of an aspartate general base in ketosteroid isomerase (KSI) by exogenous rescue. In addition to removing the general base, we systematically mutated nearby residues and probed each mutant with a series of carboxylate bases of similar pKa but varying size. Our results underscore the need for extensive and multifaceted variation to assess and minimize steric and positioning effects and determine effective molarities that estimate catalytic contributions. We obtained consensus effective molarities of ∼5 × 10(4) M for KSI from Comamonas testosteroni (tKSI) and ∼10(3) M for KSI from Pseudomonas putida (pKSI). An X-ray crystal structure of a tKSI general base mutant showed no additional structural rearrangements, and double mutant cycles revealed similar contributions from an oxyanion hole mutation in the wild-type and base-rescued reactions, providing no indication of mutational effects extending beyond the general base site. Thus, the high effective molarities suggest a large catalytic contribution associated with the general base. A significant portion of this effect presumably arises from positioning of the base, but its large magnitude suggests the involvement of additional catalytic mechanisms as well.

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.

A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s(-1)) accounts for less than 1% of the wild-type activity (k cat = 1100 ± 30 s(-1)) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s(-1), respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s(-1) and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys(104)-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

One step affinity recovery of 3α-hydroxysteroid dehydrogenase from cloned Escherichia coli.

3α-Hydroxysteroid dehydrogenase (3α-HSD), from Comamonas Testosterone, catalyze reversibly the oxidoreduction of 3α-hydroxyl groups of the steroid hormones. The gene encoding 3α-HSD (hsdA) from Comamonas Testosterone was expressed in Escherchia coli BL21 (DE3). A protocol for recovering 3α-HSD based on affinity strategy was designed and employed. Deoxycholic acid was chosen as the affinity ligand, and it was linked to Sepharose 4B with the aid of the spacers as cyanuric chloride and ethanediamine. With this specific affinity medium, the enzyme recovery process consisted of only one chromatography step to capture 3α-HSD. The target protein, analyzed on HPLC Agilent SEC-5 column, was of 94% pure among the captured protein, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 8.8% of crude extracted proteins; the recovery yield of 3α-HSD was 73.2%. 3α-HSD was revealed as a non-covalent homodimer with molecular mass of ∼56kDa by 15.0% SDS-PAGE analysis and SE-HPLC analysis. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 4.5µg/g medium and 21.3mg/g medium, respectively.

Isolation and identification of a repressor TetR for 3,17ß-HSD expressional regulation in Comamonas testosteroni.

Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17ß-Hydroxysteroid dehydrogenase (3,17ß-HSD) from C. testosteroni is a key enzyme in steroid degradation. Understanding the mechanism of 3,17ß-HSD gene (ßhsd) induction may help us to elucidate its complete molecular regulation. Sequencing the C. testosteroni ATCC11996 genome lead us to identify the tetR (522 bp) downstream of ßhsd. Two repeat sequences (RS; 13 bp), that are separated to each other by 1661 bp, were found upstream of ßhsd. A bioinformatic analysis revealed that TetR family proteins act as transcriptional repressors which are sensitive to environmental signals. Since, C. testosteroni responds to environmental steroid induction and upregulates steroid catabolic genes, we hypothesized that TetR might act in C. testosteroni as repressor for ßhsd expression. The tetR was cloned into different plasmids, including an EGFP reporter system, for functional characterization and/or overexpression. The data indicate that, indeed, TetR acts as a repressor for 3,17ß-HSD expression. Testosterone in turn, which is known to induce ßhsd expression, could not resolve TetR repression. To further substantiate TetR as repressor for ßhsd expression, a tetR gene knock-out mutant of C. testosteroni was generated. TetR gene knock-out mutants showed the same basal low level of ßhsd expression as the C. testosteroni wild type cells. Interestingly, testosterone induction leads to a strong increase in ßhsd expression, especially in the tetR gene knock-out mutants. The result with the knock-out mutant, in principle, supports our hypothesis that TetR is a repressor for ßhsd expression, but the exact role of testosterone in this context remains unknown. Finally, it turned out that TetR is obviously also involved in the regulation of the hsdA gene.

Cloning, expression and characterization of a putative 2,5-diketo-D-gluconic acid reductase in Comamonas testosteroni.

Aldo-keto reductases (AKRs) are a superfamily of soluble NAD(P)(H) oxidoreductases. The function of the enzymes is to reduce aldehydes and ketones into primary and secondary alcohols. We have cloned a 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene from Comamonas testosteroni (C. testosteroni) ATCC11996 (a Gram-negative bacterium which can use steroids as carbon and energy source) into plasmid pET-15b and over expressed in Escherichia coli BL21 (DE3). The protein was purified by His-tag Metal chelating affinity chromatography column. The 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene contains 1062 bp and could be translated into a protein of 353 amino acid residues. Three consensus sequences of the AKR superfamily are found as GxxxxDxAxxY, LxxxGxxxPxxGxG and LxxxxxxxxxDxxxxH. GxxxxDxAxxY is the active site, LxxxGxxxPxxGxG is the Cofactor-binding site for NAD(P)(H), LxxxxxxxxxDxxxxH is used for supporting the 3D structure. 2,5-diketo-D-gluconic acid reductase gene of C. testosteroni was knocked out and a mutant M-AKR was obtained. Compared to wild type C. testosteroni, degradations of testosterone, estradiol, oestrone and methyltestosterone in mutant M-AKR were decreased. Therefore, 2,5-diketo-D-gluconic acid reductase in C. testosteroni is involved in steroid degradation.