Corynebacterium glutamicum

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Structural insights into the substrate specificity of cystathionine gamma-synthase from Corynebacterium glutamicum Hye-Young Sagong, and Kyung-Jin Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02391 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 5, 2017

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Journal of Agricultural and Food Chemistry

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Structural insights into the substrate specificity of cystathionine gamma-synthase from

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Corynebacterium glutamicum

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Hye-Young Sagong and Kyung-Jin Kim*

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School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University,

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Daehak-ro 80, Buk-ku, Daegu 702-701, Korea

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*

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Kyung-Jin Kim, Ph.D.

Correspondence should be addressed:

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Structural and molecular biology Laboratory, School of Life Sciences, Kyungpook National

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University, Daehak-ro 80, Buk-ku, Daegu 702-701, Korea.

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Tel: +82-53-950-5377

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Fax: +82-53-955-5522

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E-mail: [email protected]

15 16

ACS Paragon Plus Environment


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ABSTRACT

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Cystathionine gamma synthase (MetB) condenses O-acetyl-L-homoserine (OAHS)

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or O-succinyl-L-homoserine (OSHS) with cysteine to produce cystathionine. To investigate

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the molecular mechanisms and substrate specificity of MetB from Corynebacterium

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glutamicum (CgMetB), we determined its crystal structure at a 1.5 Å resolution. The PLP

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cofactor is covalently bound to Lys204 via a Schiff base linkage in the deep cavity.

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Superposition with the structure of MetB from Nicotiana tabacum in complex with its

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inhibitor APPA revealed that Thr347 from the β10-β11 connecting loop, located at the

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entrance of the active site, is speculated to be a main contributor for the stabilization of

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acetyl-group of OAHS. Moreover, based on structural comparison of CgMetB with EcMetB

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utilizing OSHS as a main substrate, we propose that the conformation of the β10-β11

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connecting loops determines the size and shape of the acetyl- or succinyl-group binding site,

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and ultimately determines the substrate specificity of MetBs towards OAHS or OSHS.

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Keywords: Corynebacterium glutamicum, Cystathionine gamma-synthase, L-methionine


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INTRODUCTION

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Methionine is one of the essential amino acids in humans and livestock. It can be

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produced only in microorganisms and plants, and the members of methionine biosynthetic

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pathway are therefore attractive targets for designing novel antibiotics and herbicides.

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Methionine is also used as an animal feed additive for livestock. Due to increase in meat

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consumption, the global market of methionine is expected to reach USD 7.3 billion by 2022.

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Industrially, methionine is mainly produced by chemical synthesis, which produces a mixture

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of D- and L-methionine1. This process requires hazardous chemicals as ingredients and incurs

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additional costs to separate the racemic mixture. The increased demand for environment-

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friendly methionine based on renewable resources encourages bio-based methionine

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production. Bio-based methionine can be produced by enzymatic synthesis or by

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fermentation using microorganisms such as Escherichia coli and Corynebacterium

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glutamicum. Enzymatic synthesis of methionine exhibits high yields, but it requires

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expensive substrates2. Methionine production by fermentation utilizes microorganisms

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capable of producing amino acids, and attempts have been made to overproduce biologically

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active L-methionine3-6.

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C. glutamicum is a Gram-positive bacterium that has been widely utilized for the

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industrial production of various amino acids3, 7-8. In C. glutamicum, the biosynthetic pathway

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leading to L-methionine is derived from homoserine. The first step is the activation of

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homoserine, which is converted into O-acetyl-L-homoserine (OAHS) by homoserine O-

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acetyltransferase (MetX). In most organisms, an acetyl group activates the homoserine9,

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whereas in enterobacteria and some other organisms, a succinyl group is transferred to

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homoserine10-11. In addition, plants and some bacteria utilize a phosphate group. Activated

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homoserine can be converted into homocysteine in two different ways. The trans-sulfuration

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pathway via cystathionine utilizes cysteine as a sulfur donor, while direct-sulfuration pathway



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utilizes an inorganic sulfur source, such as hydrogen sulfide or methanethiol. C. glutamicum

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has been reported to utilize both trans- and direct-sulfuration pathways. In the trans-

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sulfuration pathway, cystathionine is produced by the condensation of OAHS with cysteine

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by cystathionine gamma-synthase (MetB) and subsequent hydrolysis of cystathionine by

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cystathionine beta-lyase yields homocysteine. In the direct-sulfuration pathway, OAHS reacts

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with free hydrogen sulfide, directly producing homocysteine. The final step is S-methylation

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of homocysteine, which is catalyzed by homocysteine S-methyltransferase (MetE/H).

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Cystathionine gamma-synthase (MetB) catalyzes the γ-replacement reaction of

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OAHS with cysteine, producing cystathionine (Fig. 1A). This protein is an attractive target

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for the development of novel antimicrobial compounds because it catalyzes the first reaction

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in the trans-sulfuration pathway, which is unique in bacteria12. MetB utilizes PLP as a

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cofactor and belongs to the γ-family of pyridoxal phosphate (PLP)-dependent enzymes. As

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microbial MetB uses acetyl- or succinyl-homoserine as an activated substrate, the substrate

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specificity of this enzyme is an important issue. Although the structural studies have been

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reported on MetBs from several microorganisms, such as Mycobacterium ulcerans13,

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Helicobacter pylori, Escherichia coli14, and Nicotiana tabacum15, detailed studies on

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substrate specificity of MetB proteins had not been reported yet. In addition, despite the

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importance of C. glutamicum as a producer of L-methionine, structural and biochemical

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studies on MetB had not been reported prior to this study.

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In this study, we determined the crystal structure of MetB from C. glutamicum

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(CgMetB) and elucidated the cofactor and substrate binding modes of the enzyme. In addition,

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this study provides structural insights into how the enzyme utilizes acetyl-homoserine instead

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of succinyl-homoserine as an activated substrate.

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MATERIALS AND METHODS

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Cloning, expression, and purification of CgMetB

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The CgMetB gene was amplified by polymerase chain reaction (PCR) using genomic

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DNA from C. glutamicum strain ATCC 13032 as a template with primers: forward, 5’-

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GCGCGCATATGTCTTTTGACCCAAACACCCAG-3’, and reverse, 5’-GCGCG GCGGCC

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GCAAGGTTATTGAGGGCCTGCTC-3’. The PCR product was then subcloned into pET30a

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(Novagen) with a 6x His tag at the C-terminus, and the resulting expression vector

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pET30a:CgmetB was transformed into the E. coli strain BL21(DE3)-T1R, which was grown

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in 1 L of LB medium containing kanamycin at 37 °C. After induction by the addition of 0.5

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mM isopropyl β-D-1-thiogalactopyranoside (IPTG), the culture medium was maintained for a

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further 21 h at 18 °C. The culture was then harvested by centrifugation at 4,000 × g for 15

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min at 4 °C. The cell pellet was resuspended in buffer A (40 mM Tris-HCl, pH 8.0) and then

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disrupted by ultrasonication. The cell debris was removed by centrifugation at 13,500 × g for

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30 min and the lysate was applied to an Ni-NTA agarose column (Qiagen, Germany). After

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washing with buffer A containing 10 mM imidazole, the bound proteins were eluted with 300

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mM imidazole in buffer A. Finally, trace amounts of contaminants were removed by size-

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exclusion chromatography by using a Sephacryl S-300 prep-grade column (320 mL, GE

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Healthcare, England) equilibrated with buffer A. All purification experiments were performed

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at 4 °C and SDS-polyacrylamide gel electrophoresis analysis of the purified proteins shows a

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single polypeptide of 41.7 kDa corresponding to the estimated molecular weight of the

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CgMetB monomer. The purified protein was concentrated to 50 mg/mL in 40 mM Tris-HCl,

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pH 8.0. Site-directed mutagenesis experiments were performed using the Quick Change site-

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directed mutagenesis kit (Stratagene, United States of America). The production and

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purification of the CgMetB mutants were carried out by the same procedure employed for the

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wild-type protein.



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Crystallization of CgMetB

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Crystallization of the purified protein was initially performed with commercially

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available sparse-matrix screens, including Index, PEG ion I and II (Hampton Research), and

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Wizard Classic I and II (Rigaku Reagents), using the hanging-drop vapor-diffusion method at

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20 °C. Each experiment consisted of mixing 1.0 µL protein solution (70 mg/ml in 40 mM

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Tris-HCl, pH 8.0) with 1.0 µL reservoir solution and then equilibrating this against 500 mL

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reservoir solution. CgMetB crystals of the best quality appeared in 13% polyethylene glycol

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3350 and 0.1 M Magnesium formate dihydrate.

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Data collection and structure determination of CgMetB.

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The crystals of CgMetB were transferred to cryoprotectant solution composed of the

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corresponding conditions described above and 30% (v/v) glycerol, fished out with a loop

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larger than the crystals, and flash-cooled by immersion in liquid nitrogen. All data were

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collected at the 7A beamline of the Pohang Accelerator Laboratory (PAL, Pohang, Korea),

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using a Quantum 270 CCD detector (ADSC, USA). The CgMetB crystals diffracted to a

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resolution of 1.5 Å. All data were indexed, integrated, and scaled using the HKL-2000

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software package16. The CgMetB crystals belonged to the space group F222 with unit cell

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parameters a = 58.57 Å, b = 149.85 Å, c = 161.86, α = β = γ = 90.0°. Assuming one molecule

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of CgMetB (41.7kDa) per asymmetric unit, the crystal volume per unit of protein mass was

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2.13 Å3 Da-1 with a solvent content of approximately 42.27%17. The structure of CgMetB was

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determined by molecular replacement with the CCP4 version of MOLREP18 using the

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structure of MetB from Mycobacterium ulcerans (PDB code 3QHX) as a search model.

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Model building was performed manually using the program WinCoot19, and refinement was

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performed with CCP4 REFMAC520. The data statistics are summarized in Table 1. The


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refined model of CgMetB was deposited in the Protein Data Bank with PDB code of 5X5H21.

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Site-directed mutagenesis and activity assay of CgMetB

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Site-specific mutations were created with the QuikChange kit (Stratagene), and

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sequencing was performed to confirm correct incorporation of the mutations. Mutant proteins

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were purified in the same manner as their wild-type. Primers used for cloning and site-

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directed mutagenesis are listed in Supplementary Table 1. The enzymatic activity of CgMetB

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was evaluated by using the continuous coupled assay involving cystathionine β-lyase (CBL)

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and lactate dehydrogenase (LDH). In this method, OAHS was condensed with cysteine by

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MetB into cystathionine, followed by the production of homocysteine, pyruvate and

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ammonium ion by CBL. Pyruvate is then converted into lactate by LDH with the oxidation of

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NADH to NAD+. The decrease of NADH is measured at 340 nm absorbance. Activity assays

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were performed at room temperature with a reaction mixture of 0.5 mL total volume. The

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reaction mixture contained 50 mM Tris-HCl, pH 8.0, 0.2 mM NADH, 0.1-10 mM cysteine,

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0.0001-1 mM OAHS, and 2.45 µM CBL, 1.45 µM LDH. For kinetic analysis of OSHS,

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0.005-2 mM OSHS was added to the same reaction mixture. The reaction was initiated by the

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addition of 24 µM wild-type or mutant CgMetB proteins.

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RESULTS AND DISCUSSION

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Overall structure of CgMetB

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To understand the molecular mechanism of CgMetB, we determined its crystal

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structure at 1.5 Å resolution (Table 1). The monomeric structure of CgMetB shows an overall

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fold similar to those of cystathionine gamma-synthases from other bacteria such as

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Mycobacterium ulcerans (MuMetB; PDB code 3QHX)13, Helicobacter pylori (HpMetB; PDB

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code 4L0O), and Escherichia coli (EcMetB; PDB code 1CS1)14 (Fig. 1B). The monomeric

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structure of CgMetB consists of three distinctive domains; N-terminal domain, PLP binding

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domain, and C-terminal domain (Fig. 1C). The N-terminal domain (NTD, Met1-Asn57) is

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composed of one α-helix and an extended N-terminal loop (ENL, Ala17-Tyr52) (Fig. 1C).

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The extended loop from NTD reaches to a neighboring subunit and participates in cofactor

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binding and formation of the active site entrance. The PLP-binding domain (PBD, Pro58-

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Thr253) exhibits a seven stranded β-sheet at the center and seven α-helices surround the

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central β-sheet (Fig. 1C). PBD mainly contributes to the binding of PLP cofactor and contains

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most of the residues involved in the enzyme catalysis. The C-terminal domain (CTD,

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Leu254-Leu386) shows a fold similar to that of PBD, although numbers of β-strands and α-

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helices forming the domain are different. A 4-stranded antiparallel β-sheet is located at the

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center of the domain with six α-helices covering both sides of the central β-sheet (Fig. 1C).

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Although the asymmetric unit contains one CgMetB molecule, and the tetrameric

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structure of the protein is generated by applying crystallographic F222 symmetry. Our size-

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exclusion chromatography result also showed the protein exist as a tetramer in solution (data

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not shown). The tetrameric structure of CgMetB is formed by the association of two tightly

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bound dimers, and the active sites of the enzyme are constituted by each active dimer. All

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three domains are involved in the dimerization of the CgMetB (Fig. 1D). For dimerization,

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ENL of NTD interacts with α12, α13, α14, and β10 of CTD from the neighboring molecule


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(Fig. 1D). In addition, four α-helices (α4, α5, α9, and α10) and three connecting loops (α3-β1,

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β6-β7, and α9-α10) from one molecule interact with those of the other dimer-related molecule

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through hydrogen bonds and hydrophobic interactions (Fig. 1D). PISA software22 calculated

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that within a dimer a 2,366.9 Å2 area of solvent-accessible interface per monomer is buried,

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and the percentage of participating residues is 18.2 %. The tetramer of CgMetB is formed by

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the symmetric association of two dimers (a dimer of dimers), and the ENLs and α10 from

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four monomers are found to be the main contributors for tetramerization. The dimer-dimer

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interactions include 52 hydrogen bonds and 12 salt bridges, indicating that the dimer-dimer

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interface is highly polarized. PISA software22 calculated that a 22,456.5 Å2 area of solvent-

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accessible interface per monomer is buried upon tetramer formation.

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Cofactor binding mode of CgMetB

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Cystathionine gamma-synthase utilizes PLP as a cofactor, and it belongs to fold type

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I (Aspartate aminotransferase family) of PLP-dependent enzymes. The PLP cofactor is

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covalently bound to Lys204 via a Schiff base linkage (Fig. 2A). The cofactor binding site is

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located at a deep cavity and formed by N-terminal ends of two α-helices (α4 and α5) and

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three connecting loops (β4-α7, β5-α8, and β6-β7). NTD from the neighboring monomer also

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contributes to the formation of cofactor binding site. Most of the residues involved in the

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stabilization of PLP are from PBD (Fig. 2B). The positively charged pyridine nitrogen of PLP

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is stabilized by a hydrogen bond with the carboxylate group of Asp179, increasing the

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electrophilic character of the cofactor (Fig. 2B). In addition, the hydroxyl group of the

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pyridine ring interacts with Asn154 through a hydrogen bond (Fig. 2B). The pyridine ring is

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fixed by van der Waals interactions with Thr181 and Ser201 on the side facing the protein

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and with Tyr107 on the other side (Fig. 2B). Interactions between aromatic residues and the

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pyridine ring are observed in most of PLP-dependent enzymes, possibly increasing the



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electron sink properties of the cofactor. The phosphate group of PLP is the main anchor of the

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cofactor binding. The phosphate moiety of PLP is mainly stabilized by hydrogen bonds with

201

the side-chains of Ser201 and Thr203 and the main-chain nitrogen atoms of Gly82 and Met83

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(Fig. 2B). In addition, the side-chains of Tyr52 and Arg54 from the neighboring monomer

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stabilize the phosphate moiety of PLP by forming hydrogen bonds (Fig. 2B).

204 205

Substrate binding mode of CgMetB

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To date there has not been reported a crystal structure of cystathione gamma-synthase

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family protein in complex with its substrate. To investigate the substrate binding mode of

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CgMetB, we first tried to determine the crystal structure of the protein in complex with the

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OAHS, which turned out not to be successful. However, we could identify the substrate

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binding mode of CgMetB by superposing the structure of the protein with that of MetB from

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Nicotiana tabacum (NtMetB; PDB code 1I41)15 in complex with its inhibitor, DL-E-2-amino-

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5-phosphono-3-pentenoic acid (APPA). The substrate binding site is formed by the CTD and

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ENL of the neighboring monomer (Fig. 3A). By analogy with the structure of NtMetB in

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complex with APPA, the α-carboxyl group of OAHS might be oriented in hydrogen bonding

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distances to Asn154, Ser332, and Arg364. The side-chains of Asn154 and Arg364, and main-

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chain of Ser332 stabilize the α-carboxyl group of substrate (Fig. 3B). The acetyl-group of

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OAHS might be located at the entrance of the active site that is constructed by the CTD loops

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(connecting loops of α12-β10 and β10-β11) and ENL from the other monomer. The position

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of the phosphate moiety of APPA seems to be similar to that of the carbonyl moiety of the

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acetyl-group. In the structure of NtMetB in complex with APPA, the phosphate moiety of

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APPA is stabilized by the side chains of Glu107 and Tyr111 through hydrogen bonds (Fig.

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3B). However, it was observed that the Tyr111 is substituted by Val55 in CgMetB, and Glu51

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and Val55 are located distal from the acetyl-group. Instead of these residues, Glu331 from the


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α12-β10 connecting loop and Thr347 from the β10-β11 connecting loop might be involved in

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the stabilization of the carbonyl moiety of OAHS (Fig. 3B). The methyl moiety of acetyl

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group seems to be stacked around Val55 through hydrophobic contact (Fig. 3B). After OAHS

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binds to the enzyme, the second substrate L-cysteine has to bind to a different binding site.

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The OAHS would occupy the main part of the binding pocket and then L-cysteine binds to

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the active site entrance. We investigated a second potential binding site that is

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electrostatically optimally designed for binding the L-cysteine, and expect that L-cysteine

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might be docked with the α-amino and α-carboxyl groups through hydrogen bond interactions

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with putative recognition functions Glu51 and Glu331, and Arg112, respectively (Fig. 3C).

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To verify the residues involved in the substrate binding mode of CgMetB, we

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performed site-directed mutagenesis experiments based on our structural observations of the

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protein and compared the enzyme activities of the mutants with that of the wild-type protein.

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We mutated residues that are speculated to be involved in the stabilization of the substrate

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OAHS and L-cysteine to alanine, and compared the MetB activities of the mutants with that

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of the wild-type. The CgMetBE51A, CgMetBV55A, CgMetBN154A, CgMetBE331A, and

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CgMetBS332A mutants showed only 50% MetB activities, and the CgMetBR112A mutant

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exhibited 20% MetB activity, as compared with that of the wild-type (Fig. 3D). These results

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indicate that the residues Glu51, Val55, Asn154, Glu331, Ser332, and Arg112 contribute to

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the binding of the OAHS and cysteine substrates. In addition, CgMetBT347A and CgMetBR364A

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showed almost complete loss of activity (Fig. 3D). Thus, we propose that the Thr347 and

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Arg364 residues are the main contributors for the substrate binding.

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Substrate specificity of MetB proteins

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Microbial cystathionine gamma-synthases condense acetyl- or succinyl-homoserine

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with L-cysteine to produce cystathionine. It has been reported that CgMetB uses only OAHS



249

as an activated substrate but not O-succinyl-L-homoserine (OSHS)23, however, EcMetB is

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known to utilize OSHS as a main substrate24-25. In order to determine the substrate specificity

251

of CgMetB, we performed kinetic analysis for OAHS, OSHS, and L-cysteine. For all three

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substrates, the kcat values were similar each other with 0.0106 min-1, 0.0115 min-1, and

253

0.0114 min-1 for OAHS, OSHS, and L-cysteine, respectively (Fig. 4A, B, C). However, the

254

Km values for these substrates were quite different each other with 1.30 µM, 10.2 µM, and

255

242.3 µM for OAHS, OSHS, and L-cysteine, respectively (Fig. 4A, B, C). The Km values for

256

OSHS was 8-fold higher than that for OAHS. The results indicate that CgMetB has much

257

higher affinity for OAHS and utilizes it as a main substrate. To investigate structural features

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that determine substrate specificities of MetBs, we superimposed the monomeric structure of

259

CgMetB with that of EcMetB. The overall structures of these proteins were quite similar to

260

each other, with R.M.S.D. values for all atoms of 1.26 Å. However, structural differences

261

were observed at the substrate binding site, especially, the conformation of the β10-β11

262

connecting loop in CgMetB which exhibits noticeable structural differences compared with

263

that of the corresponding loop in EcMetB (Fig. 4D). Compared to that of EcMetB, the loop in

264

CgMetB is located closer to the PLP binding site, which consequently makes smaller

265

substrate binding site of CgMetB than that of EcMetB (Fig. 4E, F). Because OAHS has two

266

less carbons than OSHS, the substrate binding pocket of CgMetB should be smaller than that

267

of EcMetB using OSHS as a substrate. Based on these structural observations, we suggest

268

that the conformation of the β10-β11 connecting loop in MetB determines the size of

269

substrate binding pocket and ultimately determines the substrate specificity of MetB.

270

Interestingly, the Thr347 residue, whose mutation to alanine showed almost complete loss of

271

activity (Fig. 3D), is located on the β10-β11 connecting loop in CgMetB, which supports our

272

suggestion that the conformation of the β10-β11 connecting loop is a determinant of substrate

273

specificities of MetBs.


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In summary, we reported the crystal structure of MetB using OAHS as a substrate,

275

CgMetB, and provided structural insight into the substrate specificity of MetB proteins. We

276

also proposed that the conformation of the substrate binding site and its size might determine

277

the substrate specificity of the MetB proteins. These structural information might

278

significantly influence L-methionine biosynthesis.



279

AUTHOR INFORMATION

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Corresponding Author

281

Telephone: +82-53-950-5377. Fax: +82-53-955-5522. E-mail: [email protected]

282 283

Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

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This work was supported by C1 Gas Refinery Program through the National

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Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future

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Planning (NRF-2016M3D3A1A01913269), and was also supported by the New &

290

Renewable Energy Core Technology Program of the Korea Institute of Energy Technology

291

Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade,

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Industry & Energy, Republic of Korea (20153030091360).

293 294 295 296

Author contribution K-J.K. designed the project. H-Y.S. performed the experiments. H-Y.S and K-J.K. wrote the paper.

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Table 1. Data collection and refinement statistics of CgMetB. CgMetB PDB code Data collection Wavelength (Å) Cell dimensions (a, b, c; α, β, γ) (Å; °) Space group Resolution range (Å) Rsym (%) I / σI Completeness (%) Redundancy Refinement Resolution (Å) No. reflections Rwork / Rfree No. atoms Protein Ligand/ion Water B-factors (Å2) Protein Ligand/ion Water B from Wilson plot (Å2) R.m.s. deviations Bond lengths (Å) Bond angles (°) a

5X5H 0.97934 58.57, 149.85, 161.86; 90.0, 90.0, 90.0 F222 50.00-1.51 (1.54-1.51) 7.8 (30.5) 42.7 (16.0) 99.3 (98.6) 6.6 (5.0)

50.00-1.51 55876 14.8/17.2 3211 2922 35 254 18.0 17.9 23.0 28.4 15.4 0.027 2.465

The numbers in parentheses are statistics from the highest resolution shell.


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FIGURE LEGENDS

Figure 1. Overall structures of CgMetB. (A) Methionine biosynthetic pathway highlighting the enzyme reaction of CgMetB. (B) Amino acid sequence alignment of the MetB proteins. Identical and highly conserved residues are presented in red and blue colored characters, respectively. Secondary structure elements are shown and labeled based on the structure of CgMetB. Residues involved in the substrate and cofactor binding are marked with red and blue colored triangles, respectively. The extended N-terminal loop is indicated by the letters ENL. Cg, Mu, Hp, and Ec represent MetB from Corynebacterium glutamicum, Mycobacterium ulceran, Helicobacter pylori, and Escherichia coli, respectively. (C) Monomeric structure of CgMetB. A monomeric CgMetB is shown as a cartoon diagram. NTD, PBD, and CTD are distinguished with green, cyan, and salmon colors, respectively. PLP molecule bound in the enzyme is shown as a magenta sphere. (D) Tetrameric structure of CgMetB. The tetrameric structure of CgMetB is presented as a cartoon diagram. In the active dimer, one monomer is shown with a color scheme in (C) and NTD, PBD, CTD from one monomer are distinguished with yellow, light-blue, and orange colors, respectively. The other active dimer is presented in gray color. The right-side figure is a 90° rotation in the vertical direction from the left-side figure.

Figure 2. Cofactor binding mode of CgMetB. (A) Electron density map of PLP. The Fo-Fc eletron density map of the bound PLP is shown as a blue mesh and contoured at 3.5 σ. (B) Cofactor binding mode of CgMetB. The CgMetB structure in complex with PLP is presented with a surface model with colors of cyan, salmon, yellow, and light-blue for the PBD and CTD of one monomer and the NTD and PBD of the other monomer, respectively. The residues involved in the PLP binding are shown as stick



models and labeled. The hydrogen bonds involved in the PLP binding are shown as redcolored dotted lines.

Figure 3. Substrate binding mode of CgMetB. (A) A surface model of the substrate binding site of CgMetB. The CgMetB structure in complex with PLP is presented with a surface model with colors of cyan, salmon, and yellow for the PBD and CTD of one monomer and the NTD of the other monomer, respectively. PLP and APPA are shown as stick model with magenta and pink colors, respectively. (B) Substrate binding mode of CgMetB. The dimeric structure of CgMetB in complex with PLP is superimposed with that of NtMetB in complex with its inhibitor APPA. Monomer I and II of CgMetB are distinguished with green and cyan colors, and monomer I and II of NtmetB are presented with colors of light-blue and wheat. The residues involved in the substrate binding are shown as stick models and labeled. The hydrogen bonds involved in the APPA binding are shown as red-colored dotted lines. (C) Cysteine binding site of CgMetB. Putative cysteine binding site of CgMetB is shown as cartoon diagram with a color scheme in (A). The residues speculated to be involved in the cysteine binding are shown as stick models and labeled. (D) Site directed mutagenesis of CgMetB. Residues involved in substrate binding are replaced by alanine residues. The relative activity of recombinant mutant proteins are measured and compared with that of wild-type CgMetB. Each experiment was performed in triplicate.

Figure 4. Substrate specificity of CgMetB. (A) (B) (C) Enzyme kinetics of CgMetB. Michaelis-Menten equation-based plot of reaction velocity versus substrate concentrations. Various concentrations of OAHS (0.0001-1 mM) (A), OSHS (0.005-2 mM) (B), and L-cysteine (0.1-10 mM) (C) were used. Each experiment


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was performed in triplicate. (D) Superimposition of the MetB structures. The monomeric structures of CgMetB and EcMetB are superimposed and shown as a cartoon diagram. CgMetB and EcMetB are distinguished with different colors of green and orange, respectively. The bound PLP and APPA were presented as a stick model with magenta and pink colors, respectively. The β10-β11 connecting loops are indicated with black dotted circles and labeled. (E) Comparsion of the conformation of substrate binding site of MetBs. The substrate binding sites of CgMetB and EcMetB are superimposed and shown as a cartoon diagram with a color scheme in (D). (F) A surface model of the substrate binding site of CgMetB and EcMetB. The structures of CgMetB and EcMetB are presented with a surface model. The β10-β11 connecting loops are indicated with yellow dotted circles.



Table of Contents Graphic


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Figure 1 600x761mm (96 x 96 DPI)





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NTD (Mol I) PBD (Mol I)

PBD (Mol II)

CgMetB

NTD (Mol II) CTD (Mol II)

CTD (Mol I)

Mol III

EcMetB

Mol IV PLP


Corynebacterium glutamicum

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