The Dual-Specific Active Site of 7,8-Diaminopelargonic Acid Synthase and the Effect of the R391A Mutation

Eliot, Andrew C.; Sandmark, J.; Schneider, Günter; Kirsch, Jack F.

doi:10.1021/bi026339a

Cited by 36 publications

(41 citation statements)

References 36 publications

(44 reference statements)

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“…The Michaelis-Menten parameters, K m and k cat , for the DAPA synthase reaction are in broad agreement with those for the corresponding E. coli enzyme (9). However, the K m of the M. tuberculosis DAPA synthase for KAPA (2.83 mM) is slightly higher than that of the E. coli enzyme (1.2 mM) (19), but similar to the B. subtilis (1 -5 mM) enzyme (16) and lower than that of the B. divaricatum (69 mM) enzyme (18).…”

Section: Kinetic Characterization Of Dapa Synthasesupporting

confidence: 68%

“…The general mechanism of an aminotransferase half reaction is proposed to proceed from internal aldimine (l max * 430 nm) via external aldimine (l max * 430 nm), quinonoid intermediate (l max * 490 nm) to the formation of ketimine or PMP form (l max * 335 nm) of the enzyme. However, unlike the E. coli enzyme (9), no transient absorbance at 487 nm corresponding to the quinonoid intermediate was observed. Observation of an isosbestic point at 372 nm also suggests the absence of any other absorption bands apart from the 420 nm and 335 nm bands.…”

Section: Spectral Characterization Of Dapa Synthasementioning

confidence: 81%

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Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

Bhor

Dev

Vasanthakumar

et al. 2006

IUBMB Life (International Union of Biochemistry and Molecular B

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SummaryThe indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 + 0.11 M 71 s 71 . The K m values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 mM and 308.28 mM, respectively whereas the V max and k cat values for the enzyme are 0.02074 mmoles/min/ml and 0.003 s 71 , respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme. IUBMB Life, 58: 225-233, 2006

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Section: Kinetic Characterization Of Dapa Synthasesupporting

confidence: 68%

Section: Spectral Characterization Of Dapa Synthasementioning

confidence: 81%

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

Bhor

Dev

Vasanthakumar

et al. 2006

IUBMB Life (International Union of Biochemistry and Molecular B

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“…Assuming the substrate/enzyme molar ratio used in our in vitro experiments to be saturating for the enzyme, the k cat value for the ester hydrolysis by BioH can be minimally estimated to be 60 s −1 . This value is orders of magnitude greater than those reported for the downstream enzymes of the pathway (20)(21)(22)(23). This mismatch of BioH catalytic activity with the other enzymes of the pathway and with the physiological need for only extremely modest amounts of biotin may indicate that the enzyme has not yet been fully integrated into E. coli biotin synthesis.…”

Section: Discussionmentioning

confidence: 77%

“…1), may be an intrinsically poor catalyst (4). This lack of catalytic efficiency is also characteristic of the downstream enzymes of the pathway (20)(21)(22)(23), and has been attributed to the traces of biotin required for cellular growth (24). To verify this premise, we sought to determine the kinetic constants for the esterase reaction mediated by BioH and attempted several in vitro assays, all of which were unsuccessful.…”

Section: Discussionmentioning

confidence: 99%

Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis

Agarwal

Lin²,

Lukk

et al. 2012

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

128

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Although the pimeloyl moiety was long known to be a biotin precursor, the mechanism of assembly of this C7 α,ω-dicarboxylic acid was only recently elucidated. In Escherichia coli, pimelate is made by bypassing the strict specificity of the fatty acid synthetic pathway. BioC methylates the free carboxyl of a malonyl thioester, which replaces the usual acetyl thioester primer. This atypical primer is transformed to pimeloyl-acyl carrier protein (ACP) methyl ester by two cycles of fatty acid synthesis. The question is, what stops this product from undergoing further elongation? Although BioH readily cleaves this product in vitro, the enzyme is nonspecific, which made assignment of its physiological substrate problematical, especially because another enzyme, BioF, could also perform this gatekeeping function. We report the 2.05-Å resolution cocrystal structure of a complex of BioH with pimeloyl-ACP methyl ester and use the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester.cofactor biosynthesis | esterase | protein-protein interaction R ecent work delineated the assembly pathway of the enigmatic pimeloyl moiety of biotin. Labeling studies in Escherichia coli had shown that pimelate, a C7 α,ω-dicarboxylic acid, is made by head-to-tail incorporation of three intact acetate units with one of the carboxyl groups being derived from CO 2 (1, 2). The differing origins of the carboxyl groups indicated that free pimelate was not a synthetic intermediate. The acetate incorporation pattern was consistent with use of the synthetic pathway that produces the usual monocarboxylic fatty acids. However, synthesis of a dicarboxylic acid using the fatty acid synthetic pathway appeared precluded by the strongly hydrophobic active sites of the fatty acid synthetic enzymes (3), which seemed unlikely to tolerate the charged carboxyl group in place of the usual terminal methyl group. The solution to this conundrum was provided by the characterization of two enzymes, BioC and BioH, which do not directly catalyze pimelate synthesis but instead allow fatty acid synthesis to assemble the pimelate moiety (4). Such circumvention of the specificity of normal fatty acid synthesis begins by BioC (an Omethyltransferase) conversion of the ω-carboxyl group of malonylacyl carrier protein (ACP) to a methyl ester using S-adenosyl-Lmethionine (SAM) as a methyl donor (Fig. 1). Conversion to a methyl ester neutralizes the negative charge and provides a methyl carbon that mimics the methyl ends of normal fatty acyl chains. The malonyl-ACP methyl ester can now enter the fatty acid synthetic pathway where it is condensed with malonyl-ACP by a 3-oxoacyl-ACP synthase in a decarboxylating Claisen reaction to give 3-oxoglutaryl-ACP methyl ester. The methyl ester shielding allows the 3-oxo group to be processed to a methylene group by the standard fatty acid reductase-dehydratase-reductase reaction sequence. The resulting glutaryl-ACP methyl ester would then be elongated to the C7 species, and another r...

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“…Nevertheless, kinetic parameters (k 0.5 and k cat values for the plant bifunctional enzyme) compared well with those (K m and k cat values) of bacterial monofunctional enzymes (Krell and Eisenberg, 1970;Gibson et al, 1995;Yang et al, 1997;Eliot et al, 2002;Mann and Ploux, 2006;Dey et al, 2010) (Table 1). A noticeable exception concerned the k cat value for plant overall reaction (which reflects DAPA-AT activity) that was found to be one order of magnitude lower than that for bacterial DAPA-AT (Eliot et al, 2002;Mann and Ploux, 2006).…”

Section: Biochemical Characterization Of Dapa-at and Dtbs Reactionsmentioning

confidence: 76%

Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase–Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis

et al. 2012

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Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling.

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The Dual-Specific Active Site of 7,8-Diaminopelargonic Acid Synthase and the Effect of the R391A Mutation

Cited by 36 publications

References 36 publications

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis

Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase–Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis

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