Crystal structure of Escherichia coli alkanesulfonate monooxygenase SsuD.

The FMNH(2)-dependent alkanesulfonate monooxygenase SsuD catalyzes the conversion of alkanesulfonates to the corresponding aldehyde and sulfite. The enzyme allows Escherichia coli to use a wide range of alkanesulfonates as sulfur sources for growth when sulfate or cysteine are not available. The structure of SsuD was solved using the multiwavelength anomalous dispersion method from only four ordered selenium sites per asymmetric unit (one site per 20,800 Da). The final model includes 328 of 380 amino acid residues and was refined to an R-factor of 23.5% (R(free)=27.5%) at 2.3A resolution. The X-ray crystal structure of SsuD shows a homotetrameric state for the enzyme, each subunit being composed of a TIM-barrel fold enlarged by four insertion regions that contribute to intersubunit interactions. SsuD is structurally related to a bacterial luciferase and an archaeal coenzyme F(420)-dependent reductase in spite of a low level of sequence identity with these enzymes. The structural relationship is not limited to the beta-barrel region; it includes most but not all extension regions and shows distinct properties for the SsuD TIM-barrel. A likely substrate-binding site is postulated on the basis of the SsuD structure presented here, results from earlier biochemical studies, and structure relatedness to bacterial luciferase. SsuD is related to other FMNH(2)-dependent monooxygenases that show distant sequence relationship to luciferase. Thus, the structure reported here provides a model for enzymes belonging to this family and suggests that they might all fold as TIM-barrel proteins.

Regulation of the degradative pathways from 4-toluenesulphonate and 4-toluenecarboxylate to protocatechuate in Comamonas testosteroni T-2.

Comamonas testosteroni T-2 was grown in salts medium containing intermediates of the established, inducible degradative pathway(s) for 4-toluenesulphonate/4-toluenecarboxylate. The specific activity or, if appropriate, the specific expression of pathway enzymes or their components was constant throughout growth and decreased only slowly in the stationary phase. It was found that the 4-toluenesulphonate methyl-monooxygenase system and 4-sulphobenzyl alcohol dehydrogenase (with 4-sulphobenzaldehyde dehydrogenase) were always co-induced, with similar ratios of their activities during growth with 4-toluenesulphonate, 4-toluenecarboxylate and 4-sulphobenzoate. We presume these enzymes to be co-expressed from one regulatory unit. The ratio of activities of the terephthalate 1,2-dioxygenase system to those of (1R,2S)-dihydroxy-1,4-dicarboxy-3,5-cyclohexadiene dehydrogenase was also constant, and present only during growth with 4-toluenecarboxylate or terephthalate. We presume these two enzymes to be co-expressed from a different regulatory unit. The oxygenase component of 4-sulphobenzoate 3,4-dioxygenase (PSBDOS) was expressed at high levels in most growth conditions examined, the exception being with 4-toluenecarboxylate as carbon source. However, no expression of a specific reductase activity linked to synthesis of the oxygenase of PSBDOS could be detected. The PSBDOS was thus active in vivo solely under conditions where the 4-toluenesulphonate methyl-monooxygenase system was also present, whose reductase is active with the oxygenase of the 4-sulphobenzoate 3,4-dioxygenase system in vitro, and, apparently, in vivo. The synthesis of PSBDOS is thus under the control of a third regulatory unit.

Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains.

Sequence comparison of pseudomurein endoisopeptidases PeiW encoded by the defective prophage PsiM100 of Methanothermobacter wolfeii, and PeiP encoded by phage PsiM2 of Methanothermobacter marburgensis, revealed that the two enzymes share only limited similarity. Their amino acid sequences comprise an N-terminal domain characterized by the presence of direct repeats and a C-terminal domain with a catalytic triad C-H-D as in thiol proteases and animal transglutaminases. Both PeiW and PeiP catalyze the in vitro lysis of M. marburgensis cells under reducing conditions and exhibit characteristics of metal-activated peptidases. Optimal temperature and pH were determined to be 63 degrees C and 6.4 for His-tagged PeiP and 71 degrees C and 6.4 for His-tagged PeiW, respectively. Database search results suggest that PeiW and PeiP are the first two experimentally identified members of a novel family of proteases in a superfamily of archaeal, bacterial, and eukaryotic protein homologs of animal transglutaminases.

Biosynthesis of Proline.

Proline was among the last biosynthetic precursors to have its biosynthetic pathway unraveled. This review recapitulates the findings on the biosynthesis and transport of proline. Glutamyl kinase (GK) catalyzes the ATP-dependent phosphorylation of L-glutamic acid. Purification of γ-GK from Escherichia coli was facilitated by the expression of the proB and proA genes from a high-copy-number plasmid and the development of a specific coupled assay based on the NADPH-dependent reduction of GP by γ-glutamyl phosphate reductase (GPR). GPR catalyzes the NADPH-dependent reduction of GP to GSA. Site directed mutagenesis was used to identify residues that constitute the active site of E. coli GK. This analysis indicated that there is an overlap between the binding sites for glutamate and the allosteric inhibitor proline, suggesting that proline competes with the binding of glutamate. The review also summarizes the genes involved in the metabolism of proline in E. coli and Salmonella. Among the completed genomic sequences of Enterobacteriaceae, genes specifying all three proline biosynthetic enzymes can be discerned in E. coli, Shigella, Salmonella enterica, Serratia marcescens, Erwinia carotovora, Yersinia, Photorhabdus luminescens, and Sodalis glossinidius strain morsitans. The intracellular proline concentration increases with increasing external osmolality in proline-overproducing mutants. This apparent osmotic regulation of proline accumulation in the overproducing strains may be the result of increased retention or recapture of proline, achieved by osmotic stimulation of the ProP or ProU proline transport systems. A number of proline analogs can be incorporated into proteins in vivo or in vitro.

Sequence variation in dichloromethane dehalogenases/glutathione S-transferases.

Dichloromethane dehalogenase/glutathione S-transferase allows methylotrophic bacteria to grow with dichloromethane (DCM), a predominantly man-made compound. Bacteria growing with DCM by virtue of this enzyme have been readily isolated in the past. So far, the sequence of the dcmA gene encoding DCM dehalogenase has been determined for Methylobacterium dichloromethanicum DM4 and Methylophilus sp. DM11. DCM dehalogenase genes closely related to that of strain DM4 were amplified by PCR and cloned from total DNA from 14 different DCM-degrading strains, enrichment cultures and sludge samples from wastewater treatment plants. In total, eight different sequences encoding seven different protein sequences were obtained. Sequences of different origin were identical in several instances. Sequence variation was limited to base substitutions; strikingly, 16 of the 19 substitutions in the dcmA gene itself encoded amino acids that were different from those of the DM4 sequence. The kinetic parameters k(cat) and K:(m), the pH optimum and the stability of representative DCM dehalogenase variants were investigated, revealing minor differences between the properties of DCM dehalogenases related to that from strain DM4.

Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates.

A 5 kb region upstream of katA at 82 degrees on the Bacillus subtilis chromosome contains five ORFs organized in an operon-like structure. Based on sequence similarity, three of the ORFs are likely to encode an ABC transport system (ssuBAC) and another to encode a monooxygenase (ssuD). The deduced amino acid sequence of the last ORF (ygaN) shows no similarity to any known protein. B. subtilis can utilize a range of aliphatic sulfonates such as alkanesulfonates, taurine, isethionate and sulfoacetate as a source of sulfur, but not when ssuA and ssuC are disrupted by insertion of a neomycin-resistance gene. Utilization of aliphatic sulfonates was not affected in a strain lacking 3'-phosphoadenosine 5'-phosphosulfate (PAPS) sulfotransferase, indicating that sulfate is not an intermediate in the assimilation of sulfonate-sulfur. Sulfate or cysteine prevented expression of beta-galactosidase from a transcriptional ssuD::lacZ fusion. It is proposed that ssuBACD encode a system for ATP-dependent transport of alkanesulfonates and an oxygenase required for their desulfonation.

Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates.

Pseudomonas aeruginosa PAO1 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates. During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of 10 sulfate starvation-induced (SSI) proteins was strongly up-regulated, as observed by two-dimensional protein electrophoresis of total cell extracts. A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response. One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specific antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent. To study the role that cysteine biosynthetic intermediates play in the synthesis of these proteins in vivo, we isolated mini-Tn5 transposon mutants of P. aeruginosa with insertions in the cysN and cysI genes, which encode subunits of ATP-sulfurylase and sulfite reductase, respectively. These two genes were cloned and sequenced. cysI showed high similarity to the cognate gene in Escherichia coli, whereas cysN encoded a 69.3 kDa protein with two domains corresponding to the E. coli CysN and CysC proteins. Sulfate no longer repressed synthesis of the SSI proteins in cysN mutants, but repression was restored by sulfite; in the cysI mutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis. This suggests that there are at least two independent corepressors of the sulfate starvation response in this species.

Chloromethane-induced genes define a third C1 utilization pathway in Methylobacterium chloromethanicum CM4.

Methylobacterium chloromethanicum CM4 is an aerobic alpha-proteobacterium capable of growth with chloromethane as the sole carbon and energy source. Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of chloromethane and the vitamin B12-mediated transfer of the methyl group of chloromethane to tetrahydrofolate. Three genes located near cmuA and cmuB, designated metF, folD and purU and encoding homologs of methylene tetrahydrofolate (methylene-H4folate) reductase, methylene-H4folate dehydrogenase-methenyl-H4folate cyclohydrolase and formyl-H4folate hydrolase, respectively, suggested the existence of a chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4. Hybridization and PCR analysis indicated that these genes were absent in Methylobacterium extorquens AM1, which is unable to grow with chloromethane. Studies with transcriptional xylE fusions demonstrated the chloromethane-dependent expression of these genes. Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with chloromethane. The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the Methylobacterium promoters described thus far. As shown previously for purU, inactivation of the metF gene resulted in a CM4 mutant unable to grow with chloromethane. Methylene-H4folate reductase activity was detected in a cell extract of strain CM4 only in the presence of chloromethane but not in the metF mutant. Taken together, these data provide evidence that M. chloromethanicum CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with chloromethane.

Transformation of isopropylamine to L-alaninol by Pseudomonas sp. strain KIE171 involves N-glutamylated intermediates.

Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or L-alaninol [S-(+)-2-amino-1-propanol] as the sole carbon source, but not with D-alaninol. To investigate the hypothesis that L-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and L-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to L-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of gamma-glutamyl-isopropylamide from isopropylamine, ATP, and L-glutamate, was shown to be catalyzed by IpuC, a gamma-glutamylamide synthetase. gamma-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding gamma-glutamyl-L-alaninol [gamma(L-glutamyl)-L-hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate L-alaninol and to regenerate L-glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters. Presumably, L-alaninol is oxidized by an alcohol dehydrogenase to yield L-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either L-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.