Proline Reduction Research Articles

Exploring the Proline-Dependent Conformational Change in the Multifunctional PutA Flavoprotein by Tryptophan Fluorescence Spectroscopy

The multifunctional PutA flavoprotein regulates proline utilization in Escherichia coli by switching from a cytosolic DNA-binding protein to a membrane-bound enzyme with proline dehydrogenase (PRODH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. The transformation of PutA from a transcriptional repressor of the proline utilization (put) regulon to a peripheral membrane associated enzyme is mediated by a proline-dependent conformational change. Previously, limited proteolysis of PutA indicated that the conformational change involves a flexible domain of unknown function (residues 141-262) which is nearby the FAD-binding and PRODH active sites (residues 263-610). Here, we extend our understanding of the proline-dependent conformational change in PutA by investigating the intrinsic Trp fluorescence spectroscopic properties of a truncated PutA protein which contains residues 86-601 (PutA86-601) and only four Trp residues. The addition of proline to wild-type PutA86-601 decreases Trp fluorescence by 36%, indicating a substantial conformational change. An apparent rate constant of 0.59 +/- 0.06 s(-)(1) was determined for the fluorescence change by stopped-flow fluorescence measurements. The limiting rate constant for proline reduction of the FAD cofactor in PutA is 133 +/- 6 s(-)(1), demonstrating that FAD reduction precedes the conformational transition observed by Trp fluorescence. The nonreducing ligand l-tetrahydro-2-furoic acid mimics the decrease in Trp fluorescence induced by proline, indicating that both FAD reduction and ligand binding contribute to the observed conformational change in PutA86-601. W194 and W211, which are located in the flexible domain, were replaced by Phe in the PutA86-601 mutants W194F, W211F, and W194F/W211F to determine which residue is involved in the observed fluorescence change. Analysis of the PutA86-601 mutants indicated that W211 is the primary molecular marker of the conformational change caused by proline. Altogether, this work shows that the switching of PutA from a transcriptional repressor to a membrane-bound protein involves W211 in a flexible domain near the PRODH active site and occurs on a time scale that is >10-fold slower than the turnover number of PutA.

Characterization of a Bifunctional PutA Homologue fromBradyrhizobium japonicumand Identification of an Active Site Residue that Modulates Proline Reduction of the Flavin Adenine Dinucleotide Cofactor

PutA is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. In this work, we have begun to assess differences between bifunctional and trifunctional PutA enzymes by examining the PutA protein from Bradyrhizobium japonicum (BjPutA). Primary structure analysis of BjPutA shows it lacks the DNA-binding domain of E. coli PutA (EcPutA). Consistent with this prediction, purified BjPutA does not exhibit DNA-binding activity in native gel mobility shift assays with promoter regions of the putA gene from B. japonicum. The catalytic and redox properties of BjPutA were characterized and a reduction potential (E(m)) value of -0.132 V (pH 7.5) was determined for the bound FAD/FADH(2) couple in BjPutA that is significantly more negative ( approximately 55 mV) than the E(m) for EcPutA-bound FAD. The more negative E(m) value thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated, suggesting lipids influence the FAD redox environment. Accordingly, an E(m) value of -0.114 V (pH 7.5) was determined for BjPutA-bound FAD in the presence of polar lipids. The molecular basis for the lower reduction potential of FAD in BjPutA relative to EcPutA was explored by site-directed mutagenesis. Amino acid sequence alignment between BjPutA and EcPutA indicates only one difference in active site residues near the isoalloxazine ring of FAD: Val402 in EcPutA is substituted at the analogous position in BjPutA with Ala310. Replacement of A310 by Val in the BjPutA mutant A310V raised the reduction potential of bound FAD relative to wild-type BjPutA to an E(m) value of -0.09 V (pH 7.5). The >40-mV positive shift in the potential of the BjPutA mutant A310V suggests that the corresponding Val residue in EcPutA helps poise the FAD redox potential for thermodynamically favored proline reduction thereby allowing EcPutA to be efficiently regulated by proline availability. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation indicating further that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues.

Proline biosynthesis from L-ornithine in Clostridium sticklandii: purification of delta1-pyrroline-5-carboxylate reductase, and sequence and expression of the encoding gene, proC.

Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and delta1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)(-1) and 4.3 nkat (mg protein)(-1), respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28.9 kDa as calculated from the sequence. Apparent Km values for PCA and NADH were 0.19 mM and 0.025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent Km values: 1.55 mM for proline and 10.5 mM for NAD at pH 10.0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.

Expression of mucin genes in ulcerative colitis.

Conference Article| November 01 1995 Expression of mucin genes in ulcerative colitis NEIL MYERSCOUGH; NEIL MYERSCOUGH 1Department of Medicine Labs, Bristol Royal Infirmary, Bristol BS2 8HW Search for other works by this author on: This Site PubMed Google Scholar BRYAN WARREN; BRYAN WARREN *Dept Cellular Pathology, John Radcliffe Hospital, Oxford OX3 9DU Search for other works by this author on: This Site PubMed Google Scholar MARGARET GOUGH; MARGARET GOUGH 1Department of Medicine Labs, Bristol Royal Infirmary, Bristol BS2 8HW Search for other works by this author on: This Site PubMed Google Scholar ANTHONY CORFIELD ANTHONY CORFIELD 1Department of Medicine Labs, Bristol Royal Infirmary, Bristol BS2 8HW Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (4): 536S. https://doi.org/10.1042/bst023536s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation NEIL MYERSCOUGH, BRYAN WARREN, MARGARET GOUGH, ANTHONY CORFIELD; Expression of mucin genes in ulcerative colitis. Biochem Soc Trans 1 November 1995; 23 (4): 536S. doi: https://doi.org/10.1042/bst023536s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

The Fate of the Carboxyl Oxygens during D-Proline Reduction by Clostridial Proline Reductase

D-Proline is converted to 5-amino valeric acid by D-proline reductase. This conversion involves the reductive cleavage of the α-carbon-nitrogen bond. We have examined the fate of the carboxyl oxygen atoms during conversion of D-proline to δ-NH2-valeric acid. 18O atoms from the carboxyl group of D-proline are not lost during conversion to product. In contrast, in the conversion of glycine to acetyl phosphate by glycine reductase a carboxyl oxygen atom is lost to solvent. An intermediate acyl-enzyme is found during the reduction of glycine. We conclude that the reduction of proline proceeds without the formation of an acyl enzyme intermediate.

Proline reduction byClostridium sporogenesis coupled to vectorial proton ejection

The reduction of proline by Clostridium sporogenes NCIB8053 is coupled to transmembrane proton translocation in an uncoupler-sensitive fashion (and might therefore conserve free energy). This finding serves to explain the increase in the growth yield of this organism when proline is added to a defined growth medium containing glucose as the catabolic substrate.

Proline reduction by Clostridium sporogenes is coupled to vectorial proton ejection

The reduction of proline by Clostridium sporogenes NCIB8053 is coupled to transmembrane proton translocation in an uncoupler-sensitive fashion (and might therefore conserve free energy). This finding serves to explain the increase in the growth yield of this organism when proline is added to a defined growth medium containing glucose as the catabolic substrate.

Stickland reactions of dental plaque.

Dental plaque samples from monkeys (Macaca fascicularis) were shown to contain proline reduction activity in coupled Stickland reactions with other amino acids and also with certain end products of bacterial glucose metabolism. The unusually high concentration of bound and free proline in the oral environment may be of importance in both the production of base and in the removal of acid from the tooth surface after dietary carbohydrate ingestion.

Inhibition by glycine of the catabolic reduction of proline in Clostridium sticklandii: Evidence on the regulation of amino acid reduction

AbstractThe growth of Clostridium sticklandii on the substrate pair L‐alanine‐L‐proline (reductant‐oxidant each 40 mM) in a medium containing 2 g/l yeast extract was completely inhibited by equimolar amounts of glycine, although glycine itself should be used as oxidant by the cells. The effect of glycine was the same, whether L‐alanine, L‐arginine, or L‐serine were used as reducdants. Performance of the growth experiments in media of high osmolarity excluded the possibility that the inhibition effected by glycine was caused by the synthesis of defective cell wall peptidoglycan. In cell‐free extracts an inhibition of L‐proline reduction by glycine was observed that did not belong to anyone of the known types of kinetic inhibition. It depended upon the presence of a functioning glycine‐reducing enzyme system, besides glycine itself, and was lost after the purification of D‐proline reductase. It was concluded from these results that a protein, besides glycine, participated in the inhibition of L‐proline reduction. The regulatory implications of the inhibition for the energy metabolism of C. sticklandii are discussed.

Utilization of collagenous by-products from the meat packing industry: production of single-cell protein by the continuous cultivation of Bacillus megaterium.

The conditions for continuous cultivation of Bacillus megaterium on a collagen-derived substrate (SP-100) were determined. The optimum conditions of temperature, pH, and dilution rate were 34 C, pH 7.0, and 0.25/hr, respectively. Increasing the substrate concentration in plain tap water resulted in proportional increases in the productivity of cell mass from 0.6 g per liter per hr at 1% substrate to 1.8 g per liter per hr at 10% substrate; however, the protein content of the biomass decreased from 60 to 36%, and the protein yield decreased from 91 to 50% at substrate concentrations of 1 and 10%, respectively. These effects (decreases) were reversed up to 7.5% substrate by mineral supplementation of the medium. The productivity of biomass increased from 0.6 to 1.9 per liter per hr; the protein content of the biomass, from 43 to 54%; and the protein yield, from 60 to 93%, respectively, as the substrate concentration (with mineral supplementation of the medium) was increased from 1 to 7.5%. Spent medium could be refortified and recycled as often as five times. The amino acids in the substrate protein appeared to be utilized for growth and metabolism more or less uniformly. Analysis of the B. megaterium biomass indicated considerable enrichment of the essential amino acids and reduction of proline, glycine, and hydroxyproline as compared to the collagen-derived substrate. The Protein Efficiency Ratios obtained on the collagen-derived substrate (SP-100) and on the B. megaterium biomass, expressed as percentages of the casein reference protein, were 14 and 74%, respectively. Thus, considerable improvement in nutritional value was effected by bacterial conversion of the collagen-derived substrate into single-cell protein.

Utilization of Collagenous By-Products from the Meat Packing Industry: Production of Single-Cell Protein by the Continuous Cultivation of Bacillus megaterium

The conditions for continuous cultivation of Bacillus megaterium on a collagen-derived substrate (SP-100) were determined. The optimum conditions of temperature, pH, and dilution rate were 34 C, pH 7.0, and 0.25/hr, respectively. Increasing the substrate concentration in plain tap water resulted in proportional increases in the productivity of cell mass from 0.6 g per liter per hr at 1% substrate to 1.8 g per liter per hr at 10% substrate; however, the protein content of the biomass decreased from 60 to 36%, and the protein yield decreased from 91 to 50% at substrate concentrations of 1 and 10%, respectively. These effects (decreases) were reversed up to 7.5% substrate by mineral supplementation of the medium. The productivity of biomass increased from 0.6 to 1.9 per liter per hr; the protein content of the biomass, from 43 to 54%; and the protein yield, from 60 to 93%, respectively, as the substrate concentration (with mineral supplementation of the medium) was increased from 1 to 7.5%. Spent medium could be refortified and recycled as often as five times. The amino acids in the substrate protein appeared to be utilized for growth and metabolism more or less uniformly. Analysis of the B. megaterium biomass indicated considerable enrichment of the essential amino acids and reduction of proline, glycine, and hydroxyproline as compared to the collagen-derived substrate. The Protein Efficiency Ratios obtained on the collagen-derived substrate (SP-100) and on the B. megaterium biomass, expressed as percentages of the casein reference protein, were 14 and 74%, respectively. Thus, considerable improvement in nutritional value was effected by bacterial conversion of the collagen-derived substrate into single-cell protein.