Crystal Structure of IlvC, a Ketol-Acid Reductoisomerase, from Streptococcus Pneumoniae

Abstract

Biosynthesis of branched-chain amino acids (BCAAs), including isoleucine, leucine and valine, is required for survival and virulence of a bacterial pathogen such as Streptococcus pneumoniae. IlvC, a ketol-acid reductoisomerase (E.C. 1.1.1.86) with NADP(H) and Mg²⁺ as cofactors from the pathogenic Streptococcus pneumoniae (SpIlvC), catalyzes the second step in the BCAA biosynthetic pathway. To elucidate the structural basis for the IlvC-mediated reaction, we determined the crystal structure of SpIlvC at 1.69 Å resolution. The crystal structure of SpIlvC contains an asymmetric dimer in which one subunit is in apo-form and the other in NADP(H) and Mg²⁺-bound form. Crystallographic analysis combined with an activity assay and small-angle X-ray scattering suggested that SpIlvC retains dimeric arrangement in solution and that D83 in the NADP(H) binding site and E195 in the Mg²⁺ binding site are the most critical in the catalytic activity of SpIlvC. Crystal structures of SpIlvC mutants (R49E, D83G, D191G and E195S) revealed local conformational changes only in the NADP(H) binding site. Taken together, our results establish the molecular mechanism for understanding functions of SpIlvC in pneumococcal growth and virulence.

1. Introduction

Branched-chain amino acids (BCAAs), referring to isoleucine, leucine and valine, are essential amino acids in humans and are required for the survival and virulence of bacterial pathogens. The biosynthetic pathway of BCAAs is present in bacteria, algae, plants, fungi and archaea, but not in mammals, thereby rendering enzymes of this pathway to be good targets for intervention [1]. IlvX family proteins, exhibiting ketol-acid reductoisomerase (KARI; EC 1.1.1.86) activity, catalyze the second step in the BCAA pathway [2]. KARIs catalyze the conversion of acetohydroxy acids such as 2-acetolactate or 2-aceto-2-hydroxybutyrate to dihydroxy valerates including 2,3-dihydroxy-3-isovalerate or 2,3-dihydroxy-3-methylvalerate (Scheme 1) ¹.

In terms of cofactor requirements, KARIs promote alkyl migration and reduction in the presence of NADP(H) and Mg²⁺ [3]. Although KARI structures from some bacteria and plants have been determined, only one structure from Escherichia coli is available in both apo and holo forms [4,5].

The BCAA pathways are critical for the survival and virulence of pathogenic bacteria. For instance, the BCAA biosynthetic pathway of pathogenic Actinobacillus pleuropneumoniae is involved in swine pneumonia [6]. A. pleuropneumoniae ilvI mutant showed defects in BCAA synthesis, inhibited growth and reduced virulence in swine. Another KARI family, such as IlvE, a branched-chain amino acid aminotransferase, from the oral cavity pathogen Streptococcus mutans, is involved in acid tolerance, as well as the BCAA pathway [7].

Streptococcus pneumoniae is a pathogenic Gram-positive bacterium that is causing a global health problem [8]. This pathogen causes pneumonia, bacteraemia, meningitis, and osteomyelitis [8,9]. The ilvC mutant from Streptococcus pneumoniae reportedly leads to a reduction of its survival [10]. Despite the involvement of the S. pneumoniae IlvC (SpIlvC) of the BCAA pathway in pneumococcal diseases, the molecular reaction mechanism of SpIlvC is not well understood. To elucidate the structural basis of SpIlvC function, we attempted to crystallize SpIlvC. By combination of crystallographic and small-angle X-ray scattering (SAXS) analyses of the SpIlvC structure, along with biochemical activity assays, we provide a molecular framework for understanding the function of SpIlvC.

2. Materials and Methods

2.1. Materials

2.1.1. DNA Cloning and Mutagenesis

Genomic DNA of S. pneumoniae strain D39 was generously provided by Dong-Kwon Rhee (Sungkyunkwan University, Suwon, Republic of Korea). DNA polymerase (Pfu polymerase) was purchased from Solgent Inc (Daejeon, Republic of Korea); restriction enzymes (BamHI and XhoI), calf intestinal alkaline phosphatase (CIP), T4 DNA ligase, and DpnI enzyme for site-directed mutagenesis were purchased from New England Biolab (Ipswitch, USA).

2.1.2. Protein Expression and Purification

Luria–Bertani (LB) medium was purchased from the USB corporation (Cleveland, OH, USA); isopropyl-1-thio-β-D-galatopyranoside (IPTG) from GeneDepot (Katy, TX, USA); Tris-HCl and NaCl from Noble BioSciences Industries (Hwaseong, Republic of Korea); imidazole from SigmaAldrich(St. Louis, MO, USA); Ni-NTA agarose resin from Qiagen (Hilden, Germany); a Superdex 200 HiLoad prep-grade column for size-exclusion chromatography from GE HealthCare (Chicago, IL, USA); and Amicon 3 kDa-centrifugal filters for concentrating protein solutions from MilliporeSigma (Burlington, NJ, USA).

2.1.3. Crystallization

Crystallization screening kits, such as Crystal Screen I/II, were purchased from Hampton Research (Aliso Viejo, CA, USA) and Wizard I/II and Cryo I/I from Molecular Dimensions (Sheffield, UK).

2.1.4. Activity Assay

2-Acetolactate was synthesized. NADPH was purchased from SigmaAldrich ( St. Louis, MO, USA).

2.2. Methods

2.2.1. DNA Cloning, Protein Expression and Purification of IlvC from S. Pneumoniae

Gene encoding SpIlvC was amplified by polymerase chain reaction (PCR) using genomic DNA of the S. pneumonaie strain D39 as a template. The amplified DNA was digested with BamHI and XhoI. Digested DNA was inserted into the BamHI/XhoI-digested expression vector parallel-His2 [11], which contained a 6xHis-tag and a TEV protease recognition site. The parallel-His2-SpIlvC plasmid was transformed to E. coli BL21(DE3) cells for protein expression. Cells were grown at 310 K in 20 mL of LB medium supplemented with 100 μg/mL ampicillin. After 16–21 h, SpIlvC cells were transferred to 2 L of LB medium supplemented with 200 μg/mL ampicillin, and further grown at 310 K until OD₆₀₀ reached 0.6–0.8. The cells were induced for protein expression by addition of 0.5 mM (final concentration) isopropyl-β-d-thiogalactopyranoside, and subsequently grown at 293 K for an additional 18–20 h.

The cells were harvested by centrifugation at 4000 rpm for 12 min at 277 K. The resulting cell pellets were resuspended in buffer A (50 mM Tris-HCl pH 7.5 and 150 mM NaCl), and homogenized by ultrasonication 2s/2s pulse for 30 min. Supernatant containing the soluble His₆-SpIlvC was collected by centrifugation at 13,000 rpm for 1 h at 277 K and subsequently loaded onto Ni-NTA Agarose resin. After incubating and washing with buffer B (50 mM Tris-HCl pH 7.5, 0.5 M NaCl and 20 mM imidazole), the protein was eluted in buffer C (50 mM Tris-HCl pH 7.5, 0.5 M NaCl and 0.3 M imidazole). His-tag was cleaved by GFP-TEV protease during dialysis against buffer A overnight at 277 K. The dialyzed solution supplemented with 20 mM imidazole was loaded onto Ni-NTA Agarose resin to remove the His₆-tag and GFP-TEV protease. Flow-through was collected and the cleaved SpIlvC was further purified on a Superdex-200 size-exclusion column pre-equilibrated with buffer A. Fractions containing SpIlvC were pooled and concentrated using a 3 kDa centrifugal filter. The quality of the purified SpIlvC was checked by SDS-PAGE analysis (Supplementary materials, Figure S1).

2.2.2. Site-Directed Mutagenesis

Site-directed mutagenesis was performed based on the protocol for QuikChange site-directed mutagenesis kit (Agilent). Briefly, pHis2-SpIlvC plasmid was amplified with designed mutant primers by PCR. The resulting solution was treated by addition of 1 μL DpnI to remove the parental plasmid by incubation of 1.5 h at 310 K. The resulting mutant plasmid was transformed to E. coli strain DH5α. A single colony was inoculated in 10 mL of LB medium supplemented with 100 μg/mL ampicillin and grown at 310 K for 16–18 h. Cells were harvested by centrifugation at 4000 rpm for 12 min at 277 K. Identities of mutants were verified by DNA sequencing.

2.2.3. Crystallization

Crystallization screening for SpIlvC wild-type (WT) and its mutants was carried out by micro-batch and hanging-drop vapor-diffusion methods. Crystallization results for SpIlvC WT were described previously [12]; therefore, they are not shown in this study. Crystallization conditions of SpIlvC mutants (R49E, D83G, D191G and E195S) are summarized in

Table 1. Crystallization conditions of SpIlvC mutants.

Parameter	R49E	D83G	D191G	E195S
Method	Hanging Drop Vapor Diffusion
Plate type	24-Well Plate
Temperature (K)	288	295	288	295
Protein concentration	8.2 mg mL−1	9 mg mL−1	12 mg mL−1	7.25 mg mL−1
Buffer composition of protein solution	50 mM Tris-HCl pH 7.5, 150 mM NaCl	50 mM Tris-HCl pH 7.5, 150 mM NaCl	50 mM Tris-HCl pH 7.5, 150 mM NaCl	50 mM Tris-HCl pH 7.5, 150 mM NaCl
Composition of reservoir solution	0.1 M HEPES pH 7.5, 0.1 M NaCl, 1.8 M ammonium sulfate, 25% glycerol	0.1 M HEPES pH 7.5, 0.1 M NaCl, 1.8 M ammonium sulfate	0.1M HEPES pH 7.5, 0.1 M NaCl, 1.5 M ammonium sulfate, 15% glycerol	0.1 M Tris-HCl pH 8.0, 0.1 M NaCl, 1.6 M ammonium sulfate
Volume and ratio of drop	1 μL protein solution with 1 μL reservoir solution	1 μL protein solution with 1 μL reservoir solution	1 μL protein solution with 1 μL reservoir solution	1 μL protein solution with 1 μL reservoir solution
Volume of reservoir	500 μL	500 μL	500 μL	500 μL

.

2.2.4. Crystallographic Data Processing and Structure Determination

Dagger-shaped crystals appeared in 3–5 days. The crystals were transferred to a cryoprotectant solution containing 30% glycerol. Diffraction data were collected on a single frozen crystal in a 100 K gaseous nitrogen stream over a range of 360° with a rotation angle per image of 1.0° at beamlines 5C and 7A in Pohang Accelerator Laboratory, Korea. Data processing and reduction were carried out using HKL-2000 [13]. The data collection and processing statistics are summarized in

Table 2. Structure refinement statistics.

Parameter	WT	R49E	D83G	D191G	E195S
Diffraction source	PAL 7A
Resolution range (Å)	41.63–1.69 (1.75–1.69)*	26.81–1.95 (2.02–1.95)	30.3–2.29 (2.37–2.29)	23.87–2.02 (2.09–2.02)	43.43–2.02 (2.1–2.02)
Space group	P212121	P212121	P21	P212121	P212121
Cell axis (Å)	69.1, 104.3, 110.9	69.5, 104.1, 111.0	71.9, 68.5, 81.2,	68.9, 104.5, 112.5	69.1, 104.4, 111.6
Cell angle (°)	90, 90, 90	90, 90, 90	90, 95, 90	90, 90, 90	90, 90, 90
Completeness (%)	98.98 (98.87)	96.1 (97.9)	99.8 (100)	98.1(100)	99.8 (97.0)
σ cut-off	0	0	0	0	0
No. of reflections, working set	86927 (8538)	54008 (5172)	33561 (3244)	49986 (4989)	50411 (4895)
No. of reflections, test set	2008 (201)	2898 (284)	1760 (170)	2686(277)	2696 (257)
Final Rcryst	0.191	0.204	0.159	0.204	0.179
Final Rfree	0.206	0.244	0.212	0.242	0.219
No. of non-Hatoms
Protein	5055	5061	9941	5413	5007
Ion	26	21	40	15	24
Ligand	90	-	-	108	96
Water	696	485	277	552	433
Total	5867	5567	10258	5818	5560
R.m.s. deviations
Bonds (Å)	0.007	0.009	0.008	0.009	0.0076
Angles (°)	1.09	1.10	1.08	1.18	1.12
Average B factors (Å2)
Protein	30.1	60.9	34.7	33.4	31.5
Ion	11.3	9.73	14.1	10.7	15.6
Ligand	16.2	-	-	1.72	1.13
Water	42.4	37.3	37.2	41.3	38.6
Ramachandran plot
Most favoured (%)	96.41	96.05	97	97.69	96.8
Allowed (%)	3.59	3.34	3	2.31	3.08

^*Values for the outer shell are given in parentheses.

. For phasing by molecular replacement (MR), an MR solution was found using PHENIX [14] with a KARI from Pseudomonas aeruginosa sharing 54% sequence identity (PDB code: 1NP3) as a search model. Iterative model building and refinement were done using PHENIX and COOT [15]. Diffraction data and coordinates have been deposited to the protein data bank with identification codes 6L2I (WT), 6L2K (R49E), 6L2S (D83G), 6L2Z (D191G) and 6L2R (E195S).

2.2.5. Reductase Activity Assay

2-Acetolactate (5.9 mM solution), a substrate, was prepared by basic hydrolysis of its O-acetyl ethyl ester according to the procedures described in [16,17] and stored at −20 °C. The reaction scheme of 2-Acetolactate is described in Supplementary Figure S2. SpIlvC (1 μM) was pre-incubated in the activity assay buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, and 350 μM NADP(H)) at 30 °C for 20 min. After pre-incubation, the 0 to 1.24 mM substrate was added to the reaction mixture and decrease in the absorbance at 340 nm was monitored on a spectrophotometer (GE HealthCare). The absorbance at 340 nm reflects the change in the concentration of NADP(H). K_m values were determined using the Michaelis–Menten equation.

2.2.6. Small-Angle X-Ray Scattering (SAXS) Measurement, Data Processing and Validation

Purified IlvC protein was concentrated to 2.94 mg/mL in buffer A, and SAXS data were obtained at beamline 4C at the Pohang Accelerator Laboratory, Korea. Buffer A was measured for subtraction of the buffer. Serially diluted (three-, nine-fold) samples were measured to ensure concentration-independent behavior of the sample in solution. The 1D SAXS profile was obtained by a home-made program at beamline 4C. The final SAXS profile was generated by merging a low-concentration profile with that of a high-concentration sample by PRIMUS [18]. The Guinier region is analyzed by AUTORG, and the pair distribution function (P(r)), D_max, and Porod volume were calculated by AUTOGNOM [19]. To avoid the ambiguity of ab initio shape determination, ten different models were generated by DAMMIF [20] and analyzed by DAMAVER to select the best model [21]. The best model—the one with the lowest NSD—was chosen for the final molecular envelope model for IlvC. The comparison of the experimental data with the calculated scattering profile of the crystal structure was performed by the FoXS server [22,23]. SAXS data processing and refinement statistics are summarized in

Table 3. Small-angle X-ray scattering data processing and analysis statistics.

Parameter	SpIlvC WT
Data collection parameters
Synchrotron beamline	PAL 4C
Beam geometry	Mica window solid cell/Oscillation capillary
Wavelength (Å)	1.24
Exposure time (min)	0.5
Concentration range (mg mL-1)	2.94
Sample parameters
Polydispersity (%, by DLS)	9.45
Structural parameters
I(0) (cm−1) [from Guinier]	7064.9 ± 16.2
Rg (Å) [from Guinier]	27.5 ± 0.1
I(0) (cm−1) [from P(r)]	7160
Rg (Å) [from P(r)]	27.9
Dmax (Å)	85.8
Porod volume estimate (Å)	100470
Software employed
Primary data reduction	In-house program at PAL 4C
Data processing	PRIMUS
Ab initio analysis	DAMMIF/DAMMIN
Validation and averaging	CRYSOL
Three-dimensional representations	PyMOL

.

3. Results

3.1. SpIlvC Reveals an Asymmetric Dimer in the Crystal

We determined the crystal structure of SpIlvC WT at a resolution of 1.69 Å. Known plant and bacterial KARI structures are reportedly categorized as classes I and II [24]. The SpIlvC structure corresponds to a class I KARI protein from Gram-positive bacteria (Supplementary Table S1). The crystal of SpIlvC contained two molecules in the asymmetric unit, consistent with homologous KARIs being dimers (Figure 1a). Interestingly, we found electron density for NADP(H) in one protomer, although we did not include NADP(H) in the crystallization conditions (Figure 1b). We infer that the NADP(H) molecule may have originated from E. coli cytoplasm. Subsequent refinement revealed that one protomer contained both magnesium ions and NADP(H), thereby constituting an asymmetric dimer in the crystal. This is in contrast to the observation that most bacterial and plant KARI structures have artificially added co-factors [25,26,27,28].

3.2. Comparison of Apo and Cofactor-Bound Structures

The overall conformation of SpIlvC remains the same regardless of the presence of cofactors such as NAPDH and Mg²⁺, as evidenced by an root mean square deviation (r.m.s.d.) of Cα atoms between the apo and holo forms of 1.0 Å. Superposition of the structures of two protomers showed larger local conformation changes in the N-terminal domain harbouring the NADP(H) binding site than in the C-terminal domain (Figure 2a). A residue-level r.m.s.d. comparison revealed that residues 50–60, corresponding to the α2 helix of the β2α2 loop showed the largest r.m.s.d. (Figure 2b). KARIs harbor a GxGxxG motif, which is part of the nucleotide binding site by phosphate-bridging interaction [24]. Ser-28 in the GxGxxG loop contacting NADP(H), exhibits a large r.m.s.d. (Figure 2b), consistent with the idea that cofactor binding causes local conformational changes [29]. These results suggest that the N-terminal domain of SpIlvC undergoes large local conformational changes upon NADP(H) and Mg²⁺ binding.

3.3. Structures of Active-Site Mutants

Analysis of residues contacting NADP(H) and Mg²⁺ identified five amino acid residues as contacting ones: Arg-49, Asp-83, Ser-53, Asp-191, and Glu-195 (Figure 3a). Structure-based multiple sequence alignment of SpIlvC and KARIs from bacteria and plants with known structures revealed that residues contacting both NADP(H) and Mg²⁺ are conserved, while the overall length of each KARI is different (Supplementary Figure S3). Since IlvC and its orthologous proteins have either reductase or isomerase activity [4], we determined the reductase activity of SpIlvC wild-type (WT) and its active-site mutants. Enzymatic activity of SpIlvC was determined by monitoring the decrease of absorbance at 340 nm characteristic of NADP(H) [4]. To investigate the effects of charge and size of the side chains of active-site residues on the kinetics of SpIlvC, we prepared 13 mutants in five residues: R49G, R49A, and R49E; S53G, S53A, S53T, and S53K; D83G; D191G and D191K; and E195A, E195S, and E195K. All the mutants exhibited significantly reduced activities compared to that of WT (Figure 3b), corroborating the structural analysis. Kinetic parameters for SpIlvC WT and mutants are summarized in

Table 4. Kinetic parameters of SpIlvC wild-type and its mutants.

Mutant	KM (μM)	kcat (s−1)	kcat/KM (μM−1s−1)a
WT	70 ± 1.21	0.10 ± 0.01	1.4 × 10−3
R49G	395 ± 57	0.16 ± 0.01	4.3 × 10−4
R49A	588 ± 194	0.18 ± 0.01	3.0 × 10−4
R49E	472 ± 134	0.18 ± 0.02	3.8 × 10−4
S53G	199 ± 34	0.11 ± 0.02	5.5 × 10−4
S53A	677 ± 61	0.17 ± 0.02	2.5 × 10−4
S53T	345± 83	0.12 ± 0.01	3.4 × 10−4
S53K	471 ± 29	0.14 ± 0.02	3.0 × 10−4
D83G	330 ± 37	0.13 ± 0.03	3.9 × 10−4
D191G	355 ± 44	0.15 ± 0.01	4.2 × 10−4
D191K	1036 ± 298	0.22 ± 0.02	2.1 × 10−4
E195A	732 ± 201	0.12 ± 0.01	1.6 × 10−4
E195S	1467 ± 142	0.25 ± 0.03	1.7 × 10−4
E195K	895 ± 185	0.18 ± 0.01	2.0 × 10−4

^aThe values are for NADP(H).

. Representative kinetic data for SpIlvC WT and selected mutants (R49E, D83G, D191G and E195S) are shown in Supplementary Figure S4. The reductase activity of SpIlvC is comparable to those of E. coli KARI and a Methanococcus aeolicus KARI mutant [25].

To obtain further structural details on the roles of active-site residues, we determined crystal structures of five mutants: two NADP(H) binding-site mutants (R49E and D83G) and two Mg²⁺ binding-site mutants (D191K and E195S) at resolutions ranging from 1.7 Å to 2.3 Å (Table 2). Superposition of structures of WT and NADP(H) binding-site mutants, such as R49E and D83G, revealed rotameric changes in residues His-31, Lys-52, Phe-54, and His-135 (Figure 4a,b). By contrast, structures of Mg²⁺ binding-site mutants, such as D191G and E195S, showed no change in rotamers of the corresponding residues in the structure of WT (Figure 4c,d). These results suggested that bulky residues in the NADP(H) binding site move to the outward positions, resulting in the NADP(H) binding pocket broadening through rotameric changes. The NADP(H) site mutants are likely to bind to NADP(H) weakly.

3.4. Solution Behaviour of SpIlvC

The crystal structure of SpIlvC revealed an asymmetric dimer (Figure 1a). To investigate the solution structure, we employed a small-angle X-ray scattering (SAXS) method. The radius of gyration (R_g) and maximal distance (D_max) of SpIlvC in solution were estimated to be 27.5 Å and 85.8 Å, respectively (Figure 5a,b), which were both consistent with the values derived from the crystal structure-R_g being 27.6 Å andsupple D_max being 89 Å. Comparison of the experimental solution scattering curve with the calculated one based on the crystal structure revealed a χ² value of 1.57 (Figure 5c). The ab initio molecular envelope derived from SAXS data appears to be well matched with the crystal structure (Figure 5d). These results support that conformation of SpIlvC remains consistent both in crystalline and solution states.

4. Discussion

We determined the crystal structures of SpIlvC wild-type and mutants to investigate the mechanistic aspects of the function of SpIlvC. The SpIlvC crystal contains an asymmetric dimer where one protomer is in the holo form, bound by cofactors such as Mg²⁺ and NADP(H), while the other is in the apo form (Figure 1). Since we did not intend to co-crystallize SpIlvC with cofactors, these ligand molecules appear to have originated from E. coli cytoplasm during expression [30]. An unnatural asymmetric dimer is not unprecedented. Unlike our structure, for other structures of KARI in complex with the cofactors, the cofactors are found in all subunits (Supplementary Table S1). Small-angle X-ray light scattering (SAXS) analysis of SpIlvC demonstrated that the structure of SpIlvC is consistent in both crystalline and solution states (Figure 5). The SAXS-derived molecular envelope does not clarify whether SpIlvC retains the asymmetric dimer, as observed in the crystal unambiguously. Nevertheless, our structure provides a rather less-represented case of an asymmetric dimer observed in a crystal.

Local conformational changes can affect the catalytic activity and cofactor binding of an enzyme. KARIs have a GxGxx(G/A)xxx(G/A) motif as a binding site for NADP(H), and Mg²⁺ is required for binding NADP(H) [3,5,25]. Structure-based multiple-sequence alignment of KARIs showed that residues constituting NADP(H) and Mg²⁺ binding sites are well conserved, while the overall length of each KARI protein is different (Supplementary Figure S3). Comparative analysis of the crystal structures of the NADP(H) binding-site mutants (R49E and D83G) and Mg²⁺ binding-site mutants (D191G and E195S) uncovered that local conformational changes are observed only in the NADP(H) binding site (Figure 4b)—His-31, Lys-52, Phe-54 and His-135—and not in the Mg²⁺ binding one (Figure 4d). These results implicate that differential rotameric changes would occur in the NADP(H) binding sites only, despite the similar detrimental effects by mutations in both NADP(H) and Mg²⁺ binding-site residues on the catalytic activity of SpIlvC.

BCAA biosynthesis is critical for survival and virulence of pneumococcal pathogens. A small amount of essential BCAA exists in a healthy pig lung and A. pleuropneumoniae, a respiratory pathogen causing swine pneumonia, seems to be responsible for such a low level of BCAA [6]. The ilvI mutant of A. pleuropneumoniae inhibits growth of the pathogen in respiratory swine. A recent physiological study reported that ilvC mutant induced defects in the BCAA pathway in S. pneumoniae [10]. The defective ilvC mutant of S. pneumoniae leads to reduced cell growth and colonization, and to increased survival of the infected rats. Given the biological and pathological importance of pneumococcal enzymes in the BCAA biosynthesis, our study discloses the mechanistic aspects of the functioning of IlvC and thereby, can serve as a starting point for understanding the effects of SpIlvC on pneumococcal virulence and physiology.