Enzymatic Activity Analysis and Catalytic Essential Residues Identification of<i>Brucella abortus</i>Malate Dehydrogenase

Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Zhang, Yuxi; Sun, Xiaoqing; Wang, Shaohui; Qiu, Xusheng; Ding, Chan; Yu, Shuang

doi:10.1155/2014/973751

Cited by 5 publications

(4 citation statements)

References 21 publications

(25 reference statements)

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“…GRP-78 is involved in protein folding and assembly [18]. MDH1 is an oxidoreductase that plays a part in tricarboxylic acid cycle [19]. CK-7 can stimulate DNA synthesis in cells [20].…”

Section: Discussionmentioning

confidence: 99%

l-Lactate Dehydrogenase B Chain Associated with Milk Protein Content in Dairy Cows

Wang

Jeon

Jung

et al. 2019

Animals

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This study aimed to explore genes associated with milk protein content in dairy cows and their relationships with l-leucine. Ten primiparous Holstein cows (93.8 ± 11.56 milking days) fed the same diet were divided into two groups depending on their milk protein contents (group High, 3.34 ± 0.10%; and group Low, 2.86 ± 0.05%). Milk epithelial cells (MECs) were isolated from the collected morning milk and differentially expressed proteins in MECs were explored by two-dimensional gel electrophoresis (2-DE). Then, the mRNA expression of these proteins was detected by real time PCR in MAC-T cells incubated with three different media named positive control (PC), negative control (NC), and l-leucine depletion (NO-leu). Results showed that ten proteins were differentially expressed in MECs from cows in group High. They included seven down-regulated ones (heat shock protein beta-1 (HSPB1), 78 kDa glucose-regulated protein (GRP-78), l-lactate dehydrogenase B chain (LDH-B), malate dehydrogenase, cytoplasmic (MDH1), annexin I (ANXA1), cytokeratin-7 (CK-7), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)), and three up-regulated ones (prohibitin (PHB), beta casein (CSN2), and alpha S1 casein (CSN1S1)). When l-leucine was depleted from the medium, not only proteins content was lowered (p < 0.05), but also the LDH-B mRNA expression was decreased in MAC-T cells (p < 0.05). In conclusion, LDH-B is negatively associated with the milk protein content of dairy cows and has a positive association with l-leucine.

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“…GRP-78 is involved in protein folding and assembly [18]. MDH1 is an oxidoreductase that plays a part in tricarboxylic acid cycle [19]. CK-7 can stimulate DNA synthesis in cells [20].…”

Section: Discussionmentioning

confidence: 99%

l-Lactate Dehydrogenase B Chain Associated with Milk Protein Content in Dairy Cows

Wang

Jeon

Jung

et al. 2019

Animals

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“…The presence of a Glu at position 42 and an Arg at position 92 (Fig 2) predicts that CBU1241 should have MDH function [28,33]. Other residues typical of the LDH and MDH superfamily involved in substrate binding (Arg98, Asn131 and Arg162) and proton transfer during catalytic action (Asp159 and His187) are conserved in CBU1241 [33,[57][58][59]. No residues uncharacteristic of an MDH were identified in CBU1241.…”

Section: The Cbu1241 Amino Acid Sequence Is Consistent With Other Malate Dehydrogenases and Cbu1241 Possesses Malate Dehydrogenase Functimentioning

confidence: 99%

Characterisation of putative lactate synthetic pathways of Coxiella burnetii

et al. 2021

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The zoonotic pathogen Coxiella burnetii, the causative agent of the human disease Q fever, is an ever-present danger to global public health. Investigating novel metabolic pathways necessary for C. burnetii to replicate within its unusual intracellular niche may identify new therapeutic targets. Recent studies employing stable isotope labelling established the ability of C. burnetii to synthesize lactate, despite the absence of an annotated synthetic pathway on its genome. A noncanonical lactate synthesis pathway could provide a novel anti-Coxiella target if it is essential for C. burnetii pathogenesis. In this study, two C. burnetii proteins, CBU1241 and CBU0823, were chosen for analysis based on their similarities to known lactate synthesizing enzymes. Recombinant GST-CBU1241, a putative malate dehydrogenase (MDH), did not produce measurable lactate in in vitro lactate dehydrogenase (LDH) activity assays and was confirmed to function as an MDH. Recombinant 6xHis-CBU0823, a putative NAD+-dependent malic enzyme, was shown to have both malic enzyme activity and MDH activity, however, did not produce measurable lactate in either LDH or malolactic enzyme activity assays in vitro. To examine potential lactate production by CBU0823 more directly, [13C]glucose labelling experiments compared label enrichment within metabolic pathways of a cbu0823 transposon mutant and the parent strain. No difference in lactate production was observed, but the loss of CBU0823 significantly reduced 13C-incorporation into glycolytic and TCA cycle intermediates. This disruption to central carbon metabolism did not have any apparent impact on intracellular replication within THP-1 cells. This research provides new information about the mechanism of lactate biosynthesis within C. burnetii, demonstrating that CBU1241 is not multifunctional, at least in vitro, and that CBU0823 also does not synthesize lactate. Although critical for normal central carbon metabolism of C. burnetii, loss of CBU0823 did not significantly impair replication of the bacterium inside cells.

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“…Thus, low pH was first studied to evaluate the FRET-Probe ICD functionality 47 . For the TSA, neutral or slightly alkaline pH was used for our two main model proteins, MDH (25 nM) and KRAS (50 nM) 48,49 , as for the ICD assay, several acidic conditions were screened. Based on the preliminary testing, pH 5 was the mildest condition inducing denaturation of these model proteins in reasonable time window.…”

Section: Denaturation Through Protonation At Low Phmentioning

confidence: 99%

Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions

Mahran,

Vello,

Komulainen

et al. 2023

Sci Rep

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Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRASG12C with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRASG12C to those reported by nucleotide exchange assay.

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Enzymatic Activity Analysis and Catalytic Essential Residues Identification ofBrucella abortusMalate Dehydrogenase

Cited by 5 publications

References 21 publications

l-Lactate Dehydrogenase B Chain Associated with Milk Protein Content in Dairy Cows

l-Lactate Dehydrogenase B Chain Associated with Milk Protein Content in Dairy Cows

Characterisation of putative lactate synthetic pathways of Coxiella burnetii

Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions

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