Structural analysis of prolines and hydroxyprolines binding to the l-glutamate-γ-semialdehyde dehydrogenase active site of bifunctional proline utilization A

https://doi.org/10.1016/j.abb.2020.108727Get rights and content

Highlights

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    Crystal structures of PutA complexed with proline stereoisomers and analogs.

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    First structure of GSALDH complexed with d-proline.

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    First structure of GSALDH complexed with hydroxyproline.

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    First structure of GSALDH complexed with NADH.

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    Inhibitors occupy the aldehyde binding site, but inhibition is not competitive.

Abstract

Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of l-proline to l-glutamate using spatially-separated proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH, a.k.a. ALDH4A1) active sites. The observation that l-proline inhibits both the GSALDH activity of PutA and monofunctional GSALDHs motivated us to study the inhibition of PutA by proline stereoisomers and analogs. Here we report five high-resolution crystal structures of PutA with the following ligands bound in the GSALDH active site: d-proline, trans-4-hydroxy-d-proline, cis-4-hydroxy-d-proline, l-proline, and trans-4-hydroxy-l-proline. Three of the structures are of ternary complexes of the enzyme with an inhibitor and either NAD+ or NADH. To our knowledge, the NADH complex is the first for any GSALDH. The structures reveal a conserved mode of recognition of the inhibitor carboxylate, which results in the pyrrolidine rings of the d- and l-isomers having different orientations and different hydrogen bonding environments. Activity assays show that the compounds are weak inhibitors with millimolar inhibition constants. Curiously, although the inhibitors occupy the aldehyde binding site, kinetic measurements show the inhibition is uncompetitive. Uncompetitive inhibition may involve proline binding to a remote site or to the enzyme-NADH complex. Together, the structural and kinetic data expand our understanding of how proline-like molecules interact with GSALDH, reveal insight into the relationship between stereochemistry and inhibitor affinity, and demonstrate the pitfalls of inferring the mechanism of inhibition from crystal structures alone.

Introduction

The enzymes of proline catabolism catalyze the 4-electron oxidation of l-proline to l-glutamate (Scheme 1) [1,2]. The first step is the oxidization of l-proline to Δ1-pyrroline-5-carboxylate (P5C), catalyzed by FAD-dependent proline dehydrogenase (PRODH). P5C forms a pH-dependent equilibrium with its hydrolysis product l-glutamate-γ-semialdehyde (GSAL). The latter is the substrate for the last enzyme of proline catabolism, GSAL dehydrogenase (GSALDH, a.k.a. ALDH4A1), which catalyzes the NAD+-dependent oxidation GSAL to l-glutamate. The enzymes PRODH and GSALDH are widely conserved in eukaryotes and bacteria. In some bacteria, PRODH and GSALDH are combined into the bifunctional enzyme known as proline utilization A (PutA) [3].

PutAs are large enzymes (>1000 residues) that catalyze the oxidation of l-proline to l-glutamate using PRODH and GSALDH active sites. The crystal structures of PutAs have revealed a conserved fold in which the two active sites are separated by ~40 Å and connected by a narrow tunnel [[4], [5], [6], [7], [8]]. The tunnel implies a substrate channeling mechanism, which has been confirmed in several PutAs [5,6,[9], [10], [11]].

Substrate inhibition is an interesting aspect of PutAs. The coupled PRODH-GSALDH activity of PutA is inhibited by l-proline, the substrate of the PRODH active site. Substrate inhibition has been observed with several PutAs, with the Ki values in the range of range of 24–263 mM [6,9,10]. Substrate inhibition likely has physiological relevance, given that the Km for proline is 7–56 mM [6,9,10]. A recent crystal structure of PutA showed that the basis of substrate inhibition is the binding of l-proline in the GSALDH site [12]. This observation is consistent with other studies showing that l-proline is an inhibitor of human GSALDH, which is a monofunctional enzyme [13]. It has been suggested that the inhibition of PutA by proline may be advantageous during osmotic stress, when bacteria need to accumulate high levels of proline rather than catabolizing it [12].

The inhibition of PutA and monofunctional GSALDH by l-proline motivated us to ask a wider molecular recognition question of whether the GSALDH active site can accommodate proline stereoisomers and analogs. Here we report the high-resolution crystal structures of PutA from Sinorhizobium meliloti (SmPutA) with five different proline molecules bound in the GSALDH active site (Scheme 2): l-proline, trans-4-hydroxy-l-proline (THLP), d-proline, cis-4-hydroxy-d-proline (CHDP), and trans-4-hydroxy-d-proline (THDP). The structures reveal a conserved mode of recognition of the carboxylate group, which results in the pyrrolidine rings of the d- and l-isomers having different orientations and different hydrogen bonding environments. Kinetic measurements suggest that hydroxyprolines are weak inhibitors of the SmPutA GSALDH domain. The structures provide insight into the recognition of proline-like compounds by GSALDH, which could be useful for developing hydroxyproline-based inhibitors of proline metabolism.

Section snippets

Materials

The following compounds were purchased from Sigma: l-proline (product number P0380), d-proline (product number 858919), trans-4-hydroxy-l-proline (THLP, product number H54409), trans-4-hydroxy-d-proline (THDP, product number 702501), cis-4-hydroxy-d-proline (CHDP, product number H5877), cis-4-hydroxy-l-proline (CHLP, product number H1637).

Crystallization

SmPutA was expressed in Escherichia coli and purified as described previously [14]. SmPutA was co-crystallized with ligands at 13 °C using the sitting-drop

Electron density for ligands bound in the GSALDH active site of SmPutA

A bifunctional PRODH-GSALDH enzyme (SmPutA) was used to determine high-resolution structures of a GSALDH active site complexed with prolines and hydroxyprolines. The PRODH and GSALDH active sites in SmPutA are separated by ~40 Å (Fig. 1A). Co-crystallization and crystal soaking experiments were performed with the compounds l-proline, d-proline, CHLP, THLP, CHDP and CHLP (Scheme 2). For all the compounds except CHLP, electron density was observed in the GSALDH active site and the compounds could

Discussion

Although the inhibition of GSALDH by l-proline has been investigated previously, to our knowledge, the larger question of how d-proline and hydroxyprolines interact with the enzyme has not. l-proline was previously found to competitively inhibit (with P5C) mammalian GSALDH with Ki of 3 mM [13]. Similarly, the product l-glutamate, as well as l-glutamate analogs, inhibit mammalian GSALDH with millimolar competitive (with P5C) Ki [13,27]. We showed here that the GSALDH active site also

Databases

Coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession codes 6X99, 6X9A, 6X9B, 6X9C, and 6X9D.

Funding

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM132640 (J J T. and D.F.B.). A.N.B. was supported by a Wayne L. Ryan Fellowship through The Ryan Foundation.

Declaration of competing interest

The authors declare no competing financial interest.

CRediT authorship contribution statement

Ashley C. Campbell: Conceptualization, Methodology, Investigation, Writing - original draft, Visualization. Alexandra N. Bogner: Investigation, Visualization. Yizi Mao: Resources. Donald F. Becker: Supervision, Project administration, Funding acquisition. John J. Tanner: Conceptualization, Writing - original draft, Writing - review & editing, Visualization, Validation, Supervision, Project administration, Funding acquisition.

Declaration of Competing interest

The authors declare no competing financial interest.

Acknowledgements

We thank J. Nix and for help with remote X-ray diffraction data collection at beamlines 4.2.2 of the Advanced Light Source. Beamline 4.2.2 of the Advanced Light Source, a DOE Office of Science User Facility under Contract No. DE-AC02-05CH11231, is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169-01.

References (35)

  • E. Inagaki et al.

    Crystal structure of Thermus thermophilus Delta1-pyrroline-5-carboxylate dehydrogenase

    J. Mol. Biol.

    (2006)
  • P. Llinas et al.

    Structural studies of human placental alkaline phosphatase in complex with functional ligands

    J. Mol. Biol.

    (2005)
  • J.J. Tanner

    Structural biology of proline catabolic enzymes

    Antioxidants Redox Signal.

    (2019)
  • J.J. Tanner

    Structural biology of proline catabolism

    Amino Acids

    (2008)
  • D. Srivastava et al.

    Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

    Proc. Natl. Acad. Sci. U.S.A.

    (2010)
  • H. Singh et al.

    Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site

    Proc. Nat. Acad. Sci. USA

    (2014)
  • D.A. Korasick et al.

    Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure

    FEBS J.

    (2017)

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