Dataset Information

Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis.

ABSTRACT: NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

SUBMITTER: Meyer T

PROVIDER: S-EPMC7465668 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Structural Characterization of an <i>S</i>-enantioselective Imine Reductase from <i>Mycobacterium Smegmatis</i>.

Meyer Timo T Zumbrägel Nadine N Geerds Christina C Gröger Harald H Niemann Hartmut H HH

Biomolecules 20200731 8

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein ...[more]

PMID: 32751900

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Project description:Mycobacterium smegmatis SigF is a group III sigma factor. Its ortholog in M. tuberculosis is reported to have role in regulation and function of cell wall components. In present study we have created an M. smegmatis M-NM-^TsigF mutant by allele exchange method. M. smegmatis sigF mutant shows non pigmented phenotype and is more sensitive to hydrogen peroxide generated oxidative stress. DNA microarray analysis of M. smegmatis wild type and M-NM-^TsigF mutant suggests that SigF in this species controls the expression of several energy and central intermediary metabolism genes along with regulation of carotenoid biosynthesis. Gene expression patterns of M.smegmatis wild type and M-NM-^TsigF mutant strains were compared at two growth stages i.e. log (OD600 ~1.4) and stationary (OD600 ~3.0). M. smegmatis strains were grown in DifcoTM MiddleBrook 7H9 broth base (BD Biosciences, Sparks, MD, USA) with 0.2% glycerol (v/v) and 0.05% Tween-80 (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% albumen dextrose catalase (BD Biosciences) (v/v) at 37 0C with continuous shaking. Total RNA was isolated using Trizole (Invitrogen) method and labelled with cyanine 3 (Cy3) as per Agilent 1-color labelling protocol (Version 5.5, February 2007). Six hundred nanograms of each Cy3 labelled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were performed using Gene Expression Hybridization kit (Agilent Technologies). Hybridization was carried out in AgilentM-bM-^@M-^Ys Surehyb Chambers at 65 0C for 16 h. The hybridized slides were washed using Agilent Gene Expression wash buffers and scanned using the Agilent Microarray Scanner G Model G2565BA at 5 micron resolution. Feature extracted data was analysed using GeneSpring GX v 7.3.1 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended one color Per Chip and Per Gene Data Transformation. Set measurements less than 0.01 to 0.01 per chip, normalize to 50th percentile per gene, and normalize to specific samples.

Project description:Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using Mycobacterium smegmatis, we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in Escherichia coli We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31.IMPORTANCE The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.

Dataset Information

Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis.

Publications

Structural Characterization of an <i>S</i>-enantioselective Imine Reductase from <i>Mycobacterium Smegmatis</i>.

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