Kaj Frank Jensen

The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase A from Lactococcus lactis

BACKGROUND . Dihydroorotate dehydrogenase (DHOD) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides. Lactococcus lactis contains two genes encoding different functional DHODs whose sequences are only 30% identical. One of these enzymes, DHODA, is a highly efficient dimer, while the other, DHODB, shows optimal activity only in the presence of an iron-sulphur cluster containing protein with which it forms a complex tetramer. Sequence alignments have identified three different families among the DHODs: the two L. lactis enzymes belong to two of the families, whereas the enzyme from E. coli is a representative of the third. As no three-dimensional structures of DHODs are currently available, we set out to determine the crystal structure of DHODA from L. lactis. The differences between the two L. lactis enzymes make them particularly interesting for studying flavoprotein redox reactions and for identifying the differences between the enzyme families. RESULTS . The crystal structure of DHODA has been determined to 2.0 resolution. The enzyme is a dimer of two crystallographically independent molecules related by a non-crystallographic twofold axis. The protein folds into and alpha/beta barrel with the flavin molecule sitting between the top of the barrel and a subdomain formed by several barrel inserts. Above the flavin isoalloxazine ring there is a small water filled cavity, completely buried beneath the protein surface and surrounded by many conserved residues. This cavity is proposed as the substrate-binding site. CONCLUSIONS . The crystal structure has allowed the function of many of the conserved residues in DHODs to be identified: many of these are associated with binding the flavin group. Important differences were identified in some of the active-site residues which vary across the distinct DHOD families, implying significant mechanistic differences. The substrate cavity, although buried, is located beneath a highly conserved loop which is much less ordered than the rest of the protein and may be important in giving access to the cavity. The location of the conserved residues surrounding this cavity suggests the potential orientation of the substrate.

Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-α-1-pyrophosphate (PRPP): Determination of cellular PRPP pools and assay of PRPP synthetase activity

Abstract Conditions are described where 5-phosphoribosyl-α-1-pyrophosphate (PRPP) can be determined by thin-layer chromatographic methods commonly used for the determination of nucleoside triphosphate pools in 32P-labeled bacteria. A two-dimensional chromatographic system is described where very small pools of PRPP (about 0.03 μmol per gram dry weight bacteria) can be determined. In a uni-dimensional chromatographic system the lower limit for detection of PRPP pools is about 0.3 μmol per gram dry weight bacteria. This uni-dimensional system offers an assay also for PRPP synthetase activity even in crude extracts using [γ-32P]ATP as a substrate. The assay is highly specific due to the chromatographic isolation of PRPP and is very sensitive due to the use of 32P labeling. The chromatographic methods for determination of PRPP pools and of activities of PRPP synthetase have been applied to the analysis of some mutants of Salmonella typhimurium and have provided results that agree well with the results obtained by conventional methods of PRPP analysis.

E. coli dihydroorotate dehydrogenase reveals structural and functional distinctions between different classes of dihydroorotate dehydrogenases.

The flavoenzymes dihydroorotate dehydrogenases (DHODs) catalyze the fourth and only redox step in the de novo biosynthesis of UMP. Enzymes belonging to class 2, according to their amino acid sequence, are characterized by having a serine residue as the catalytic base and a longer N terminus. The structure of class 2 E. coli DHOD, determined by MAD phasing, showed that the N-terminal extension forms a separate domain. The catalytic serine residue has an environment differing from the equivalent cysteine in class 1 DHODs. Significant differences between the two classes of DHODs were identified by comparison of the E. coli DHOD with the other known DHOD structures, and differences with the class 2 human DHOD explain the variation in their inhibitors.

Decreasing transcription elongation rate in Escherichia Coli exposed to amino acid starvation

The time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the ‘late’ probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth‐rate. The transcription elongation rate was c. 43 nucleotides s‐1 during exponential growth and decreased abruptly to c. 20 nucleotides s‐1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lec‐promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA’ chains were continued into full‐length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s‐1 in the unperturbed growth phase to approximately 6 aa s‐1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed.

Structure of dihydroorotate dehydrogenase B: electron transfer between two flavin groups bridged by an iron-sulphur cluster.

BACKGROUND The fourth step and only redox reaction in pyrimidine de novo biosynthesis is catalyzed by the flavoprotein dihydroorotate dehydrogenase (DHOD). Based on their sequences, DHODs are grouped into two major families. Lactococcus lactis is one of the few organisms with two DHODs, A and B, belonging to each of the two subgroups of family 1. The B enzyme (DHODB) is a prototype for DHODs in Gram-positive bacteria that use NAD+ as the second substrate. DHODB is a heterotetramer composed of two different proteins (PyrDB and PyrK) and three different cofactors: FMN, FAD, and a [2Fe-2S] cluster. RESULTS Crystal structures have been determined for DHODB and its product complex. The DHODB heterotetramer is composed of two closely interacting PyrDB-PyrK dimers with the [2Fe-2S] cluster in their interface centered between the FMN and FAD groups. Conformational changes are observed between the complexed and uncomplexed state of the enzyme for the loop carrying the catalytic cysteine residue and one of the lysines interacting with FMN, which is important for substrate binding. CONCLUSIONS A dimer of two PyrDB subunits resembling the family 1A enzymes forms the central core of DHODB. PyrK belongs to the NADPH ferredoxin reductase superfamily. The binding site for NAD+ has been deduced from the similarity to these proteins. The orotate binding in DHODB is similar to that in the family 1A enzymes. The close proximity of the three redox centers makes it possible to propose a possible electron transfer pathway involving residues conserved among the family 1B DHODs.

The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two different subunits, encoded by the pyrDb and pyrK genes, and contains FMN, FAD, and [FeS] redox centers.

The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DHOdehase B) is encoded by the pyrDb gene. However, recent genetic evidence has revealed that a co-transcribed gene, pyrK, is needed to achieve the proper physiological function of the enzyme. We have purified DHOdehase B from two strains of Escherichia coli, which harbored either the pyrDb gene or both the pyrDb and the pyrK genes of L. lactis on multicopy plasmids. The enzyme encoded by pyrDb alone (herein called the δ-enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit. The δ-enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassium hexacyanoferrate(III), and molecular oxygen as electron acceptors but could not use NAD+. The DHOdehase B purified from the E. coli strain that carried both the pyrDb and pyrK genes on a multicopy plasmid (herein called the δκ-enzyme) was quite different, since it was formed as a complex of equal amounts of the two polypeptides, i.e. two PyrDB and two PyrK subunits. The δκ-enzyme was orange-brown and contained 2 mol of FAD, 2 mol of FMN, and 2 mol of [2Fe-2S] redox clusters per mol of native protein as tightly bound prosthetic groups. The δκ-enzyme was able to use NAD+ as well as dichloroindophenol, potassium hexacyanoferrate(III), and to some extent molecular oxygen as electron acceptors for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified δ-enzyme. Based on these results and on analysis of published sequences, we propose that the architecture of the δκ-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.

Structure of the Escherichia coli pyrE operon and control of pyrE expression by a UTP modulated intercistronic attentuation.

Protein synthesis in ‘minicells’ showed that the DNA immediately preceding the pyrE gene of Escherichia coli directs the formation of considerable amounts of a polypeptide (mol. wt. approximately 30 000) of unknown function. The nucleotide sequence of this DNA revealed the existence of an open reading frame (ORF) of 238 codons that ends 68 nucleotide residues upstream to the structure start of pyrE, just prior to the GC‐rich symmetry region of a sequence with features characteristic of a rho‐independent transcription terminator. Deletion of the start of this 238 codons long ORF gene resulted in a dramatic fall in the level of pyrE expression, indicating that the two genes (ORF and pyrE) constitute an operon. S1‐nuclease digestion of RNA‐DNA hybrids revealed that both genes are transcribed from two promoters (P1 and P2) located in front of the ORF start. Furthermore, when the RNA used in these experiments was prepared from cells with different levels of pyrE expression, created by manipulations in their pyrimidine nucleotide supply, the frequency of transcription initiations at P1 and P2 was found to be constitutive, while only a pyrimidine regulated fraction of the mRNA chains reached into the pyrE gene. In vitro transcription of isolated DNA fragments showed that the mRNA chains are terminated between the ORF gene and pyrE. From these observations we conclude that pyrE expression is controlled by a UTP modulated intercistronic attentuation.

RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC.

A mutant of Salmonella typhimurium with a defect in the regulation of pyr‐gene expression was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5‐fluorouracil and 5‐fluorouridine. The mutant possesses 4‐fold elevated pools of the pyrimidine nucleoside triphosphatases, UTP and CTP. The specific activities of aspartate transcarbamylase and orotate phosphoribosyltransferase are 40‐fold and 7‐fold higher in the mutant than in the parent strain when grown in minimal media. Furthermore, the synthesis of the two enzymes in the mutant is not repressed following addition of exogenous pyrimidines. The levels of carbamoylphosphate synthase and orotidine 5′‐monophosphate decarboxylase are approximately 3‐fold enhanced, while the activities of dihydroorotase and dihydroorotate oxidase appear largely unaffected by the mutation. The mutation responsible for these effects was shown to map between two known point mutations in the rpoBC gene cluster encoding the beta and beta’ subunits of RNA polymerase. These observations indicate a regulatory function of RNA polymerase in the control of pyr‐gene expression in S. typhimurium.

Crystal structure of the phosphorolytic exoribonuclease RNase PH from Bacillus subtilis and implications for its quaternary structure and tRNA binding.

RNase PH is a member of the family of phosphorolytic 3′ → 5′ exoribonucleases that also includes polynucleotide phosphorylase (PNPase). RNase PH is involved in the maturation of tRNA precursors and especially important for removal of nucleotide residues near the CCA acceptor end of the mature tRNAs. Wild‐type and triple mutant R68Q‐R73Q‐R76Q RNase PH from Bacillus subtilis have been crystallized and the structures determined by X‐ray diffraction to medium resolution. Wild‐type and triple mutant RNase PH crystallize as a hexamer and dimer, respectively. The structures contain a rare left‐handed βαβ‐motif in the N‐terminal portion of the protein. This motif has also been identified in other enzymes involved in RNA metabolism. The RNase PH structure and active site can, despite low sequence similarity, be overlayed with the N‐terminal core of the structure and active site of Streptomyces antibioticus PNPase. The surface of the RNase PH dimer fit the shape of a tRNA molecule.

Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription.

The pyrE gene of Escherichia coli is part of an operon where it is preceded by an unknown gene (orfE) that ends 8 bp before the start of the symmetry of the UTP‐modulated pyrE attenuator. On a plasmid we have inserted this attenuator region in a synthetic cloning site early in lacZ. The resulting structure contains the lac promoter‐operator, the first few codons of lacZ, 42 bp of DNA from the orfE end, the pyrE attenuator, and an in‐frame fusion pyrE‐lacZ+. The synthetic cloning sites have been used to vary the length and reading frame of the translation that begins at the lacZ start and proceeds towards the attenuator. The effects of these variations on pyrE attenuation were determined by monitoring the synthesis of beta‐galactosidase from the pyrE‐lacZ hybrid gene in cells grown with either low or high pools of UTP. Thus, a very low level of pyrE expression was observed, regardless of UTP pool size, when the translation from the lacZ start ended 31 or 62 nucleotide residues upstream to the pyrE attenuator symmetry, but a proper UTP controlled attenuation could be established if this translation ended only 8 bp before the symmetry region of the attenuator (as the native orfE gene) or 10 bp after this structure. However, a single ‘leader peptide’ read from only frequently used codons gave a high level of pyrE expression both at high and low UTP pools. These observations indicate that the coupling between transcription and translation determines the degree of mRNA chain terminations at the pyrE attenuator.(ABSTRACT TRUNCATED AT 250 WORDS)