Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus.

Porumb, Horea; Vancea, D.; Mureşan, L; Presecan, Elena; Lascu, Ioan; Petrescu, Ioan; Porumb, Tudor; Pop, R.; Bârzu, Octavian

doi:10.1016/s0021-9258(18)61237-2

Cited by 35 publications

(5 citation statements)

References 24 publications

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“…Note that the hexameric AF2‐multimer structure prediction of BsAld also had the highest pTM and ipTM score (Figure 2b) compared to all other stoichiometries, which thus agrees with our co‐evolution analysis‐based scoring approach. This predicted structure is in accordance with a broad range of prior experimental observations in homologs (Ågren et al, 2008; Baker et al, 1998; Kuroda et al, 1990; Porumb et al, 1987; Tripathi & Ramachandran, 2008), and further exhibits high structural similarity to the M. tuberculosis holo‐Ald structure in the closed conformation (Ågren et al, 2008) (PDB: 2VHZ, TM‐score: 0.81, C α ‐RMSD: 2.03 Å).…”

Section: Resultssupporting

confidence: 91%

“…No complete experimental structure of BsAld is currently available. However, Ald from other closely related Bacilli , including Bacillus cereus , Geobacillus stearothermophilus and Lysinibacillus sphaericus , have been experimentally shown to form homo‐hexamers (Kuroda et al, 1990; Porumb et al, 1987), and X‐ray crystal structures of Ald with and without its bound substrates are available from Mycobacterium tuberculosis (Ågren et al, 2008; Tripathi & Ramachandran, 2008). In these experimental structures, Ald is arranged in a homo‐hexameric complex with threefold dihedral symmetry, and X‐ray crystal structures from the cyanobacterium Phormidium lapideum (Baker et al, 1998) and the Gram‐negative thermophile Thermus thermophilus (PDB: 2EEZ) share a highly similar structure and topology.…”

Section: Resultsmentioning

confidence: 99%

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Co‐evolution at protein–protein interfaces guides inference of stoichiometry of oligomeric protein complexes by de novo structure prediction

Kilian,

Bischofs

2023

Molecular Microbiology

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The quaternary structure with specific stoichiometry is pivotal to the specific function of protein complexes. However, determining the structure of many protein complexes experimentally remains a major bottleneck. Structural bioinformatics approaches, such as the deep learning algorithm Alphafold2‐multimer (AF2‐multimer), leverage the co‐evolution of amino acids and sequence‐structure relationships for accurate de novo structure and contact prediction. Pseudo‐likelihood maximization direct coupling analysis (plmDCA) has been used to detect co‐evolving residue pairs by statistical modeling. Here, we provide evidence that combining both methods can be used for de novo prediction of the quaternary structure and stoichiometry of a protein complex. We achieve this by augmenting the existing AF2‐multimer confidence metrics with an interpretable score to identify the complex with an optimal fraction of native contacts of co‐evolving residue pairs at intermolecular interfaces. We use this strategy to predict the quaternary structure and non‐trivial stoichiometries of Bacillus subtilis spore germination protein complexes with unknown structures. Co‐evolution at intermolecular interfaces may therefore synergize with AI‐based de novo quaternary structure prediction of structurally uncharacterized bacterial protein complexes.

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Co‐evolution at protein–protein interfaces guides inference of stoichiometry of oligomeric protein complexes by de novo structure prediction

Kilian,

Bischofs

2023

Molecular Microbiology

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“…This enzyme is a key factor in the assimilation of L-alanine as an energy source through the tricarboxylic acid cycle during sporulation (McCowen & Phibbs, 1974). Alanine dehydrogenase has been purified to homogeneity from Bacillus subtilis, B. sphaericus, and B. cereus, and its enzymological properties have been elucidated (Yoshida & Freese, 1965;Ohshima & Soda, 1979;Porumb et al, 1987). The kinetic and chemical mechanisms of the enzyme reaction have also been extensively studied .…”

mentioning

confidence: 99%

Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)+-dependent dehydrogenases

Kuroda

Tanizawa²,

Sakamoto³

et al. 1990

Biochemistry

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The gene encoding alanine dehydrogenase (EC 1.4.1.1) from a mesophile, Bacillus sphaericus, was cloned, and its complete DNA sequence was determined. In addition, the same gene from a moderate thermophile, B. stearothermophilus, was analyzed in a similar manner. Large parts of the two translated amino acid sequences were confirmed by automated Edman degradation of tryptic peptide fragments. Each alanine dehydrogenase gene consists of a 1116-bp open reading frame and encodes 372 amino acid residues corresponding to the subunit (Mr = 39,500-40,000) of the hexameric enzyme. The similarity of amino acid sequence between the two alanine dehydrogenases with distinct thermostabilities is very high (greater than 70%). The nonidentical residues are clustered in a few regions with relatively short length, which may correlate with the difference in thermal stability of the enzymes. Homology search of the primary structures of both alanine dehydrogenases with those of other pyridine nucleotide-dependent oxidoreductases revealed significant sequence similarity in the regions containing the coenzyme binding domain. Interestingly, several catalytically important residues in lactate and malate dehydrogenases are conserved in the primary structure of alanine dehydrogenases at matched positions with similar mutual distances.

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“…As mRNA is produced, translation occurs on ribosomes, incorporating intracellular LALA for 45/377 amino acid residues in ALD. Translated alanine dehydrogenase proteins (ALDN) then undergo enzyme maturation, likely consisting primarily of subunit assembly, since active ALD (ALDM) is comprised of six identical subunits (Porumb et al, 1987).…”

Section: Quantitative Analysis Of Experimental Resultsmentioning

confidence: 99%

Differences in the regulatory strategies of marine oligotrophs and copiotrophs reflect differences in motility

Noell¹,

Brennan²,

Washburn³

et al. 2022

Preprint

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Aquatic bacteria are frequently divided into the lifestyle categories oligotroph or copiotroph, reflecting adaptations to low and high nutrient availability. In aquatic ecosystems, copiotrophy is associated with chemotaxis and motility, which cells use to find and occupy high-nutrient patches. Oligotrophs have proportionately fewer transcriptional regulatory proteins than copiotrophs, and some have been shown to constitutively express genes involved in the uptake and oxidation of carbon compounds. We hypothesized that the absence of chemotaxis/motility in oligotrophs might prevent them from occupying nutrient patches long enough to benefit from transcriptional regulation. To test this hypothesis, we measured uptake and oxidation of a radiolabeled amino acid, [14C]L-alanine, by a non-motile oligotroph (Ca. Pelagibacter st. HTCC7211) and a motile copiotroph (Alteromonas macleodii st. HOT1A3). We found that L-alanine catabolism is not transcriptionally regulated in HTCC7211 but is in HOT1A3, initiating within 2.5 - 4 min, as supported by RT-qPCR experiments. L-alanine uptake in HTCC7211 was modulated within 30s by low-amplitude (2-fold) post-translational regulation, a conclusion supported by quantitative analysis with a mechanistic model. By modeling cell trajectories under a range of patch conditions, we predicted that, in most scenarios, non-motile cells spend <2 min in patches, while chemotactic/motile cells occupy patches >4 mins. We conclude that the time necessary to initiate transcriptional regulation prevents non-motile oligotrophs, which drift with currents, from benefiting from transcriptional regulation, but instead have low-amplitude post-translational regulation that can take advantage of their transient passage through a patch.

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Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus.

Cited by 35 publications

References 24 publications

Co‐evolution at protein–protein interfaces guides inference of stoichiometry of oligomeric protein complexes by de novo structure prediction

Co‐evolution at protein–protein interfaces guides inference of stoichiometry of oligomeric protein complexes by de novo structure prediction

Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)+-dependent dehydrogenases

Differences in the regulatory strategies of marine oligotrophs and copiotrophs reflect differences in motility

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