Inactivation of Malonate Semialdehyde Decarboxylase by 3-Halopropiolates:  Evidence for Hydratase Activity

Poelarends, Gerrit J.; Serrano, Héctor; Johnson, William H.; Whitman, Christian P.

doi:10.1021/bi050296r

Cited by 18 publications

(36 citation statements)

References 28 publications

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“…In this regard, the unusual reactivity of FlK allows us to explore the existence of a unique chemical species in enzyme catalysis because it strongly suggests a ketene intermediate in the FlK catalytic cycle based on similarities to related chemical model systems (38)(39)(40). Although precedence for ketene intermediates exists for enzymatic reactions with mechanism-based inhibitors or nonphysiological substrates (47)(48)(49)(50)(51)(52)(53)(54), there is not yet any experimental evidence for their involvement in normal catalytic cycles despite their rich history in organic chemistry and the strong interest in their discovery in enzyme catalysis. For example, a ketene-based mechanism has been proposed in the catalytic cycle of glycine reductase, but it represents only one of several mechanistic possibilities that remain to be distinguished experimentally (55).…”

Section: Discussionmentioning

confidence: 99%

Catalytic control of enzymatic fluorine specificity

Weeks¹,

Chang

2012

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

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The investigation of unique chemical phenotypes has led to the discovery of enzymes with interesting behaviors that allow us to explore unusual function. The organofluorine-producing microbe Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that demonstrates a surprisingly high level of discrimination for a single fluorine substituent on its substrate compared with the cellularly abundant hydrogen analog, acetyl-CoA. In this report, we show that the high selectivity of FlK is achieved through catalysis rather than molecular recognition, where deprotonation at the C α position to form a putative ketene intermediate only occurs on the fluorinated substrate, thereby accelerating the rate of hydrolysis 10 4 -fold compared with the nonfluorinated congener. These studies provide insight into mechanisms of catalytic selectivity in a native system where the existence of two reaction pathways determines substrate rather than product selection.enzyme mechanism | substrate selectivity L iving organisms have solved some of the most difficult challenges in catalysis by harnessing the exquisite selectivity and reactivity of enzyme active sites to build complex chemical behaviors (1-5). Indeed, the plasticity of enzymes toward evolution of new function has allowed life to flourish in diverse biospheres by conferring a selective advantage to hosts that can demonstrate catalytic innovation and use unusual resources. The enzymes that form the molecular basis for these chemical phenotypes are a rich source of molecular diversity and provide an experimental platform for the continual search to gain insight into the mechanisms and dynamics of protein and organismal evolution (6-9). One of the salient features of enzymes is their extremely high substrate selectivity, which allows them to choose the correct substrate over the thousands of other small molecules that are present in cells at any given time. Thus, the exploration of substrate specificity and its evolution is key for understanding both enzyme catalysis and the expansion of biodiversity at the molecular level (7)(8)(9)(10)(11)(12).In this context, a singular chemical trait found in the soil bacterium Streptomyces cattleya is its ability to catalyze the formation of carbon-fluorine bonds (13,14). Fluorine resides at one corner of the periodic table, and its unique elemental properties make it highly effective as a design element for drug discovery but also quite challenging for the synthesis of fluorinecontaining molecules (15)(16)(17). Although fluorine has emerged as a common motif in synthetic compounds, including top-selling drugs such as atorvastatin and fluticasone, it has been underexploited in nature, and only a few biogenic organofluorine compounds (<20) have been identified to date despite the thousands of known natural products containing chlorine and bromine (∼5,000) (18,19). In fact, the only fully characterized organofluorine natural products are derived from a single pathway that produces the deceptively simple poison fluoroacetate (1). T...

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Section: Discussionmentioning

confidence: 99%

Catalytic control of enzymatic fluorine specificity

Weeks¹,

Chang

2012

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

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“…12,13 Pro-1 is the sole site of modification on the protein. The crystal structure of the inactivated FG41 MSAD was determined to 2.2 Å resolution using molecular replacement with the native FG41 MSAD as the search model, and refined to R and R free values of 16.4% and 20.2%, respectively.…”

Section: Resultsmentioning

confidence: 99%

Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform Bacterium Strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

Guo¹,

Serrano²,

Poelarends

et al. 2013

Biochemistry

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Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal-ion independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide, as well as a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant. Previous studies identified Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) as critical residues in these activities. MSAD from Coryneform bacterium strain FG41 (designated FG41 MSAD) shares 38% pairwise sequence identity with the Pseudomonas enzyme including Pro-1 and Asp-37. However, Gln-73 replaces Arg-73, and the second arginine is shifted to Arg-76 by the insertion of a glycine. In order to determine how these changes relate to the activities of FG41 MSAD, the gene was cloned and the enzyme expressed and characterized. The enzyme has a comparable decarboxylase activity, but a significantly reduced hydratase activity. Mutagenesis along with crystal structures of the native enzyme (2.0 Å resolution) and the enzyme modified by a 3-oxopropanoate moiety (resulting from the incubation of enzyme and 3-bromopropiolate) (2.2 Å resolution) provided a structural basis. The roles of Pro-1 and Asp-37 are likely the same as those proposed for MSAD. However, the side chains of Thr-72, Gln-73, and Tyr-123 replace those of Arg-73 and Arg-75 in the mechanism and play a role in binding and catalysis. The structures also show that Arg-76 is likely too distant to play a direct role in the mechanism. FG41 MSAD is the second functionally annotated homologue in the MSAD family of the tautomerase superfamily and could represent a new subfamily.

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“…The key residues for these activities (based primarily on crystallographic and mutagenesis studies) are Pro-1, Asp-37, and a pair of arginines (Arg-73 and Arg-75) [44,45]. In addition, there is a hydrophobic wall (Trp-114, Phe-116, Phe-123, and Leu-128) that might facilitate decarboxylation.…”

Section: Msad From Pseudomonas Pavonaceae 170 and Coryneform Bacteriumentioning

confidence: 99%

“…In addition, there is a hydrophobic wall (Trp-114, Phe-116, Phe-123, and Leu-128) that might facilitate decarboxylation. Roles for these residues in both activities have been assigned based primarily on their positions relative to a covalent adduct ( 18 , Scheme 7) derived from the hydration of 3-chloropropiolate ( 15 ) [44]. Hydration of 15 generates an acylating agent or a ketene ( 16 or 17 , respectively) that forms a covalent adduct with Pro-1.…”

Section: Msad From Pseudomonas Pavonaceae 170 and Coryneform Bacteriumentioning

confidence: 99%

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Identification and characterization of new family members in the tautomerase superfamily: Analysis and implications

Huddleston¹,

Burks²,

Whitman³

2014

Archives of Biochemistry and Biophysics

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Tautomerase superfamily members are characterized by a β–α–β building block and a catalytic amino terminal proline. 4-oxalocrotonate tautomerase (4-OT) and malonate semialdehyde decarboxylase (MSAD) are the title enzymes of two of the five known families in the superfamily. Two recent developments in these families indicate that there might be more metabolic diversity in the tautomerase superfamily than previously thought. 4-OT homologues have been identified in three biosynthetic pathways, whereas all previously characterized 4-OTs are found in catabolic pathways. In the MSAD family, homologues have been characterized that lack decarboxylase activity, but have a modest hydratase activity using 2-oxo-3-pentynoate. This observation stands in contrast to the first characterized MSAD, which is a proficient decarboxylase and a less efficient hydratase. The hydratase activity was thought to be a vestigial and promiscuous activity. However, this recent discovery suggests that the hydratase activity might reflect a new activity in the MSAD family for an unknown substrate. These discoveries open up new avenues of research in the tautomerase superfamily.

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Inactivation of Malonate Semialdehyde Decarboxylase by 3-Halopropiolates: Evidence for Hydratase Activity

Cited by 18 publications

References 28 publications

Catalytic control of enzymatic fluorine specificity

Catalytic control of enzymatic fluorine specificity

Kinetic, Mutational, and Structural Analysis of Malonate Semialdehyde Decarboxylase from Coryneform Bacterium Strain FG41: Mechanistic Implications for the Decarboxylase and Hydratase Activities

Identification and characterization of new family members in the tautomerase superfamily: Analysis and implications

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