Abstract:
:A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alpha E-beta D) of Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH). All but one of 38 enzyme variants examined were thermally stable and had native-like hydrodynamic properties. In this sample, there was no bias detected in either the DNA or amino acid sequences encoded. We argue that the alpha E-beta D surface loop sequence is unimportant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities. The selection of NAD-dependent dehydrogenases with specificity for: malate, phenyllactate, hydroxyisocaproate and 4-phenyl-2-hydroxy-butanoate from two bsLDH libraries is described. This required a highly discriminatory screen for 2-hydroxy acid dehydrogenase activity to select enzymes which, in the absence of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition. In general the amino acid residues at positions 101 and 102 which determined substrate specificity were as expected from hydrophobic and ionic complementarity to the substrate. For example, a bsLDH variant with Asn101Va1102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate. Using molecular modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate. Similarly, nine out of ten malate dehydrogenases (MDHs) selected had an arginine residue at position 102 to complement the negatively charged carboxyl group in oxaloacetate. One, Arg101Arg102 (Kcat/Kmoxaloacetate = 1.6 x 10(6) M-1 S-1) is 25% more active than the previous best synthetic MDH. There were surprises: present understanding would not have predicted the oxaloacetate transforming activity of Ser101Leu102 or the phenylpyruvate activity of Pro101Lys102. The former is about one-third as efficient as the best malate dehydrogenase selected, whilst the latter had about one-seventh of the best phenylpyruvate dehydrogenase activity.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
el Hawrani AS,Sessions RB,Moreton KM,Holbrook JJdoi
10.1006/jmbi.1996.0626subject
Has Abstractpub_date
1996-11-22 00:00:00pages
97-110issue
1eissn
0022-2836issn
1089-8638pii
S0022-2836(96)90626-9journal_volume
264pub_type
杂志文章abstract::Human serum amyloid P component crystallizes from sodium acetate buffer (pH 5.5) in the presence of calcium and polyethylene glycol 6000, at 4 degrees C. The space group is P2(1) and the cell parameters are a = 69.0 A (1 A = 0.1 nm), b = 99.3 A, c = 96.8 A, beta = 96.1. Density considerations supported by neutron scat...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(88)90529-3
更新日期:1988-07-05 00:00:00
abstract::The HNH motif is a small nucleic acid binding and cleavage module, widespread in metal finger endonucleases in all life kingdoms. Here we studied a non-specific endonuclease, the nuclease domain of ColE7 (N-ColE7), to decipher the role of the conserved asparagine and histidine residues in the HNH motif. We found, usin...
journal_title:Journal of molecular biology
pub_type: 杂志文章
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.2001.4883
更新日期:2001-08-17 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1994.1271
更新日期:1994-04-22 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2014.02.009
更新日期:2014-05-29 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/s0022-2836(96)80018-0
更新日期:1996-11-01 00:00:00
abstract::Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(91)90258-8
更新日期:1991-04-20 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2019.01.025
更新日期:2019-03-01 00:00:00
abstract::A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitut...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1999.3445
更新日期:2000-02-04 00:00:00
abstract::Transcription activator-like effectors (TALEs) recognize DNA through repeat-variable diresidues (RVDs), and TALE-DNA interactions are sensitive to DNA modifications. Our previous study deciphered the recognition of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) by TALEs. Here, we report seven crystal struct...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2019.11.023
更新日期:2020-02-14 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2016.06.009
更新日期:2016-07-31 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(92)90881-j
更新日期:1992-12-05 00:00:00
abstract::Six mutants of Escherichia coli K12 that are sensitive to visible light have been isolated. Five of them, including an amber mutant, are defective in a gene that maps near 11 minutes on the linkage map of the chromosome, and this gene has been designated visA. The sixth mutant, which was isolated from bacteria that ca...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(91)90180-e
更新日期:1991-06-05 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/s0022-2836(03)00531-x
更新日期:2003-06-20 00:00:00
abstract::Large crystals of probable amylase/protease inhibitor-B have been grown at room temperature from ammonium sulfate solution. The crystals grow within five days to dimensions of 0.6 mm x 0.6 mm x 0.6 mm. They diffract to at least 1.7 A upon exposure to synchrotron X-rays. The crystals belong to the space group P4(1)2(1)...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1993.1027
更新日期:1993-01-05 00:00:00
abstract::The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then intera...
journal_title:Journal of molecular biology
pub_type: 杂志文章
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1997.1484
更新日期:1998-01-30 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1999.2590
更新日期:1999-03-19 00:00:00
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journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2016.11.008
更新日期:2017-02-17 00:00:00
abstract::Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of eac...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.2000.3594
更新日期:2000-03-31 00:00:00
abstract::Plasmids were constructed in which a HindIII fragment of rDNA (6.4 x 10(3) base-pairs) was inserted into vectors pGEM-1 and 2 in both orientations. The DNA insert encoded the yeast 35 S precursor rRNA beginning 180 bases upstream from the 5' end of the mature 18 S rRNA and ending 289 bases beyond the 3' end of the mat...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(90)90353-N
更新日期:1990-01-20 00:00:00
abstract::The second generation Hoechst-Bayer non-nucleoside inhibitor, HBY 097 (S-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4-dihydroqui noxalin-2(1H)-thione), is an extremely potent inhibitor of HIV-1 reverse transcriptase (RT) and of HIV-1 infection in cell culture. HBY 097 selects for unusual drug-resistance mu...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1006/jmbi.1998.2171
更新日期:1998-11-27 00:00:00
abstract::LC3-associated phagocytosis (LAP) is a novel form of non-canonical autophagy where LC3 (microtubule-associated protein 1A/1B-light chain 3) is conjugated to phagosome membranes using a portion of the canonical autophagy machinery. The impact of LAP to immune regulation is best characterized in professional phagocytes,...
journal_title:Journal of molecular biology
pub_type: 杂志文章,评审
doi:10.1016/j.jmb.2017.08.012
更新日期:2017-11-24 00:00:00
abstract::The side-chains of phenylalanine and tyrosine residues in proteins are frequently found to be involved in pairwise interactions. These occur both within repeating elements of secondary structure and in tertiary and quaternary interactions. It has been suggested that they are important in protein folding and stability,...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/0022-2836(91)90725-l
更新日期:1991-03-20 00:00:00
abstract::Here, we present the basic structural properties and the thermodynamic description of a previously unknown alkali molten globule state of horse "ferricytochrome c". Both sodium and guanidinium cations stabilize the alkali-denatured state at pH 13, presumably by a charge screening mechanism. The Na(+)-stabilized confor...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2006.09.025
更新日期:2006-12-01 00:00:00
abstract::The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/s0022-2836(02)01071-9
更新日期:2002-11-22 00:00:00
abstract::Neurodegenerative disorders associated with protein misfolding are fatal diseases that are caused by fibrillation of endogenous proteins such as α-synuclein (α-syn) in Parkinson's disease (PD) or amyloid-β in Alzheimer's disease. Fibrils of α-syn are a major pathological hallmark of PD and certain aggregation intermed...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2015.01.020
更新日期:2015-03-27 00:00:00
abstract::Transcriptional pausing by RNA polymerase has been the subject of extensive investigations in vitro, yet little is known about its occurrence and significance in vivo. The transient nature of the pausing events makes them difficult to observe inside the cell, whereas their studies in vitro by classical biochemical met...
journal_title:Journal of molecular biology
pub_type: 杂志文章
doi:10.1016/j.jmb.2005.05.052
更新日期:2005-08-05 00:00:00