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Structural basis of sequential allosteric transitions in tetrameric D-lactate dehydrogenases from three Gram-negative bacteria Nayuta Furukawa, Akimasa Miyanaga, Masahiro Nakajima, and Hayao Taguchi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00557 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018
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Biochemistry
Structural basis of sequential allosteric transitions in tetrameric D-lactate dehydrogenases from three Gram-negative bacteria Nayuta Furukawa†,‡, Akimasa Miyanaga§, Masahiro Nakajima† and Hayao Taguchi†,* From †Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, ‡Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Akiha-ku, Niigata 956-8603, and §Department of Chemistry, Tokyo Institute of Technology, 212-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan
ABSTRACT D-Lactate
dehydrogenases (D-LDHs) from Fusobacterium nucleatum (FnLDH) and E. coli
(EcLDH) exhibit positive cooperativity in substrate binding, and the Pseudomonas aeruginosa enzyme (PaLDH) shows negatively cooperative substrate binding. The apo and ternary complex structures of FnLDH and PaLDH have been determined together with the apo-EcLDH structure. The three enzymes consistently form homotetrameric structures with three symmetric axes, the P-, Q- and R-axes, unlike Lactobacillus D-LDHs, P-axis-related dimeric enzymes, although apoFnLDH and EcLDH form asymmetric and distorted quaternary structures.
The tetrameric
structure allows apo-FnLDH and EcLDH to form wide inter-subunit contact surfaces between the opened catalytic domains of the two Q-axis-related subunits in coordination with their asymmetric and distorted quaternary structures.
These contact surfaces comprise inter-subunit
hydrogen bonds and hydrophobic interactions, and likely prevent the domain closure motion in initial substrate binding.
In contrast, apo-PaLDH possesses a highly symmetrical quaternary
structure and partially closed catalytic domains favorably for initial substrate binding, and forms virtually no inter-subunit contact surface between the catalytic domains, which present their negatively charged surfaces to each other at the subunit interface.
Complex FnLDH and PaLDH
possess highly symmetrical quaternary structures with closed forms of the catalytic domains, which are separate from each other at the subunit interface. Structure-based mutations successfully converted the three enzymes to their dimeric forms, which exhibited no significant cooperativity in substrate binding.
These observations indicate that the three enzymes undergo
typical sequential allosteric transitions to exhibit their distinctive allosteric functions through the
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tetrameric structures.
Introduction NAD-Dependent L- and D-lactate dehydrogenases (L-LDH (EC 1.1.1.27), and D-LDH (EC 1.1.1.28)) catalyze the chiral specific reduction of pyruvate to L- and D-lactates, respectively, with oxidation of NADH to NAD+ at the last step of the anaerobic glycolytic pathway1.
D-LDHs are
distributed in some invertebrates2 and microorganisms3, whereas L-LDHs are more widespread in organisms including vertebrates and higher plants1,3. In spite of the similarity in their catalytic function, D-LDHs are evolutionarily separate from L-LDHs, and belong to a great D-2hydroxyacid dehydrogenase (D-2-HydDH) family4, which comprises various D-2-HydDHs, such as D-phosphoglycerate (D-PGDH)5, D-hydroxyisocaproate (D-HicDH)6, and D-glycerate7,8 dehydrogenases, and even other dehydrogenases, such as phosphite9, formate10, and L-alanine11 dehydrogenases, sharing a common protein structure12. L-LDHs are some of the extensively studied enzymes as to the structure-function relationship1,
and also some of the highly divergent enzymes in primary structure and catalytic properties. Whereas vertebrates possess L-LDH isozymes, which exhibit various catalytic properties, i.e. as to kinetic parameters such as substrate Km1, substrate specificity13, and pH-dependence14, many bacteria possess allosteric-types of L-LDHs3.
The known allosteric L-LDHs regulate their
affinity to substrates (K-type regulation) usually through fructose 1,6-bisphosphate (FBP), which drastically reduces substrate Km values, and often by substrate pyruvate, which gives sigmoidal shaped substrate saturation curves in the absence of FBP.
On the other hand, D-LDHs have been
also extensively studied in the Lactobacillus enzymes for the structure-function relationship, catalytic mechanism and substrate recognition machinery15-24.
D-LDHs
are also highly
diverged enzymes in the primary structure even in the Lactobacillus enzymes, i.e. the L. bulgalicus subs. delbruekii and L. pentosus enzymes (LdLDH and LpLDH, respectively) exhibit only 60% amino acid identity (Fig. 1).
As compared with L-LDHs, however, much less is known
about allosteric types of D-LDHs, and known Lactobacillus D-LDHs exhibit no significant allosteric properties, although Tarmy and Kaplan reported in 1967 that E. coli D-LDH (EcLDH) exhibits a significant positive cooperativity in substrate binding25.
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Biochemistry
Figure 1. Structure-based sequence alignment of representative D-LDHs.
The amino acid
sequences were aligned for seven D-LDHs from F. nucleatum (FnLDH), P. aeruginosa (PaLDH), E. coli (EcLDH), Synechocystis sp. PCC 6803 (SyLDH), A. aerolicus (AaLDH), L. delbrueckii (LdLDH), and L. pentosus (LpLDH), and D-hydroxyisocaproate dehydrogenase from Lactobacillus
casei
(LcHDH)
http://www.genome.jp/tools/clustalw/).
using
the
CLASTALW
program
(URL:
The amino acid residues of the enzymes are numbered
according to the Lactobacillus D-LDHs. The residues involved in the coenzyme binding are shown in blue.
The residues constituting the substrate binding sites are shown in red.
above the sequences indicate the secondary structural elements of apo-FnLDH.
Bars
The residues
that form the inter-subunit hydrogen bonds across the P-axis are underlined. The residues forming the inter-subunit hydrogen bonds across the Q-axis, R-axis, and both are highlighted in yellow, green, and blue, respectively.
The two hinge regions between the catalytic and NAD-binding
domains are boxed.
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Previously, we reported the catalytic properties of Fusobacterium nucleatum and Pseudomonas aeruginosa D-LDH homologs (FnLDH and PaLDH, respectively), together with those of EcLDH, demonstrating that D-LDHs from Gram-negative bacteria show a high variety in the catalytic properties26.
PaLDH exhibits small pyruvate Km (
