Altering the binding determinant on the interdigitating loop of mandelate racemase shifts specificity towards that of d-tartrate dehydratase

2022 ◽  
pp. 109119
Author(s):  
Mitesh Nagar ◽  
Joshua A. Hayden ◽  
Einat Sagey ◽  
George Worthen ◽  
Mika Park ◽  
...  
Keyword(s):  
Mandelate Racemase ◽  
Tartrate Dehydratase

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Preliminary x-ray data on crystals of mandelate racemase.

1988 ◽  
Vol 263 (19) ◽  
pp. 9268-9270
Author(s):  
D J Neidhart ◽  
V M Powers ◽  
G L Kenyon ◽  
A Y Tsou ◽  
S C Ransom ◽  
...  
Keyword(s):  
X Ray ◽  
Mandelate Racemase

Substrate spectrum of mandelate racemase

2001 ◽  
Vol 15 (4-6) ◽  
pp. 213-222 ◽  
Author(s):  
Ulfried Felfer ◽  
Ulrike T. Strauss ◽  
Wolfgang Kroutil ◽  
Walter M.F. Fabian ◽  
Kurt Faber
Keyword(s):  
Mandelate Racemase ◽  
Substrate Spectrum

Perturbing the Hydrophobic Pocket of Mandelate Racemase To Probe Phenyl Motion during Catalysis†

Biochemistry ◽  
2005 ◽  
Vol 44 (25) ◽  
pp. 9013-9021 ◽  
Author(s):  
Ferhan Siddiqi ◽  
Jennifer R. Bourque ◽  
Haiyan Jiang ◽  
Marieke Gardner ◽  
Martin St. Maurice ◽  
...  
Keyword(s):  
Hydrophobic Pocket ◽  
Mandelate Racemase

An Assay for Mandelate Racemase Using High-Performance Liquid Chromatography

1999 ◽  
Vol 269 (2) ◽  
pp. 332-336 ◽  
Author(s):  
Stephen L. Bearne ◽  
Martin St. Maurice ◽  
Mark D. Vaughan
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Mandelate Racemase

scholarly journals Attempts to develop an enzyme converting DHIV to KIV

2019 ◽  
Vol 32 (6) ◽  
pp. 261-270
Author(s):  
Kenji Oki ◽  
Frederick S Lee ◽  
Stephen L Mayo
Keyword(s):  
Protein Design ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Mandelate Racemase ◽  
Acid Dehydratase ◽  
Enolase Superfamily ◽  
Beta Elimination ◽  
Cost Efficient ◽  
Hydrolysis Activity

Abstract Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.

scholarly journals Regulation of tartrate metabolism by TtdR and relation to the DcuS–DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3632-3640 ◽  
Author(s):  
Ok Bin Kim ◽  
Julia Reimann ◽  
Hanna Lukas ◽  
Uwe Schumacher ◽  
Jan Grimpo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Promoter Region ◽  
Anaerobic Conditions ◽  
Two Component System ◽  
Two Component ◽  
Fumarate Respiration ◽  
Mutual Regulation ◽  
Genes Encoding ◽  
Type Gene ◽  
Tartrate Dehydratase

Escherichia coli catabolizes l-tartrate under anaerobic conditions to oxaloacetate by the use of l-tartrate/succinate antiporter TtdT and l-tartrate dehydratase TtdAB. Subsequently, l-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS–DcuR two-component system. Expression of the l-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of l- and meso-tartrate, and repressed by O2 and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O2, nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR–ttdA promoter region. TtdR was also required for full expression of the DcuS–DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to l-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (l- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C4-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.

Mandelate Racemase and Mandelate Dehydrogenase Coexpressed RecombinantEscherichia coliin the Synthesis of Benzoylformate

2013 ◽  
Vol 77 (6) ◽  
pp. 1236-1239 ◽  
Author(s):  
Dali LI ◽  
Zhen ZENG ◽  
Junfang YANG ◽  
Peng WANG ◽  
Lei JIANG ◽  
...  
Keyword(s):  
Mandelate Racemase
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