Mechanism of Substrate Recognition and Insight into Feedback Inhibition of Homocitrate Synthase from Thermus thermophilus

Okada, Takuya; Tanaka, Takeo; Wulandari, Asri Peni; Kuzuyama, Tomohisa; Nishiyama, Makoto

doi:10.1074/jbc.m109.086330

Cited by 32 publications

(52 citation statements)

References 43 publications

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“…Collectively, our structural and functional data demonstrate that L-lysine competes with 2-OG for binding to SpHCS, in agreement with prior studies of HCS from P. chrysogenum (10), T. thermophilus (11,12), and S. cerevisiae (9). The inherent plasticity of the active site of the enzyme enables differential recognition of 2-OG and L-lysine.…”

Section: Discussionsupporting

confidence: 76%

“…4). In very recent studies, the structure of T. thermophilus HCS in complex with L-lysine was reported (12). This enzyme displays an inhibitor binding mode that is essentially identical to that of SpHCS, illustrating structural conservation throughout evolution.…”

Section: Discussionmentioning

confidence: 99%

“…Based on these studies, two models have been proposed for the kinetic mechanism by which L-lysine inhibits HCSs. Biochemical studies of HCSs from S. cerevisiae (9), Penicillium chrysogenum (10), and Thermus thermophilus (11,12) report that L-lysine is a competitive inhibitor of 2-OG, implying that the amino acid binds within the active site of the enzyme. This model leads to a paradox in understanding how HCS can accommodate 2-OG, a dicarboxylic acid, and lysine, a basic amino acid, within the same site.…”

mentioning

confidence: 99%

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Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

Bulfer

Scott

Pillus

et al. 2010

Journal of Biological Chemistry

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The ␣-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the ␣-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by L-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with L-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the (␣/␤) 8 TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the ⑀-ammonium group of L-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the L-lysine ⑀-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of L-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.

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Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

Bulfer

Scott

Pillus

et al. 2010

Journal of Biological Chemistry

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“…Lysine at M levels inhibits HCS of T. thermophilus (8). As in the instances described above, it is possible that the growth inhibition of T. thermophilus by AEC is due to the inhibition of HCS; however, we also found that the growth of a T. thermophilus mutant possessing HCS with H72L replacement, which is feedback resistant to high concentrations of lysine (9), was inhibited by AEC with the same sensitivity as that of the wild-type strain. This observation suggests that AEC acts on a target other than HCS to inhibit the growth of T. thermophilus.…”

mentioning

confidence: 46%

Two ATP-Binding Cassette Transporters Involved in (S)-2-Aminoethyl-Cysteine Uptake in Thermus thermophilus

Kanemaru

Hasebe

Tanaka

et al. 2013

Journal of Bacteriology

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bThermus thermophilus exhibits hypersensitivity to a lysine analog, (S)-2-aminoethyl-cysteine (AEC). Cosmid libraries were constructed using genomes from two AEC-resistant mutants, AT10 and AT14, and the cosmids that conferred AEC resistance on the wild-type strain were isolated. When the cosmid library for mutant AT14 was screened, two independent cosmids, conferring partial AEC resistance to the wild type, were obtained. Two cosmids carried a common genomic region from TTC0795 to TTC0810. This region contains genes encoding an ATP-binding cassette (ABC) transporter consisting of TTC0806/TTC0795, using TTC0807 as the periplasmic substrate-binding protein. Sequencing revealed that AT14 carries mutations in TTC0795 and TTC0969, causing decreases in the thermostability of the products. TTC0969 encodes the nucleotide-binding protein of a different ABC transporter consisting of TTC0967/TTC0968/TTC0969/TTC0970 using TTC0966 as the periplasmic substrate-binding protein. By similar screening for cosmids constructed for the mutant AT10, mutations were found at TTC0807 and TTC0969. Mutation in either of the transporter components gave partial resistance to AEC in the wild-type strain, while mutations of both transporters conferred complete AEC resistance. This result indicates that both transporters are involved in AEC uptake in T. thermophilus. To elucidate the mechanism of AEC uptake, crystal structures of TTC0807 were determined in several substratebinding forms. The structures revealed that TTC0807 recognizes various basic amino acids by changing the side-chain conformation of Glu19, which interacts with the side-chain amino groups of the substrates.

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“…However, in T. thermophilus, lysine is synthesized via the ␣-aminoadipate pathway. Crystal structures of TtHCS have recently been solved (10), but a detailed study of the kinetic and/or chemical mechanism of TtHCS has not been carried out. In this article, the TtHCS has been subjected to initial rate and isotope effect studies, and the pH dependence of kinetic parameters has been measured.…”

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confidence: 99%

Kinetic and Chemical Mechanisms of Homocitrate Synthase from Thermus thermophilus

Kumar

West

Cook

2011

Journal of Biological Chemistry

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The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal-activated enzyme with either Mg 2؉ or Mn 2؉ capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with ␣-ketoglutarate (␣-Kg) binding prior to acetylCoA (AcCoA) with Mn 2؉ , whereas it is steady state random with Mg 2؉ , suggesting a difference in the competence of the E⅐Mn⅐␣-Kg⅐AcCoA and E⅐Mg⅐␣-Kg⅐AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d 3 ) in the presence of Mg 2؉ is unity (value 1.0) at low concentrations of AcCoA, whereas it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg 2؉ or Mn 2؉ as the divalent metal ion, whereas V/K ␣-Kg (with Mn 2؉ ) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as ␣-Kg. Lysine binding exhibits negative cooperativity, indicating cross-talk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.

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Mechanism of Substrate Recognition and Insight into Feedback Inhibition of Homocitrate Synthase from Thermus thermophilus

Cited by 32 publications

References 43 publications

Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

Two ATP-Binding Cassette Transporters Involved in (S)-2-Aminoethyl-Cysteine Uptake in Thermus thermophilus

Kinetic and Chemical Mechanisms of Homocitrate Synthase from Thermus thermophilus

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